Peptides for treatment of scleroderma

ABSTRACT

Peptides are provided that ameliorate one or more symptoms of scleroderma. In various embodiments, the peptides range in length from about 10 to about 30 amino acids, comprise a class A amphipathic helix, and bear at least one protecting group. The peptides are highly stable and readily administered via an oral route.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant Nos: HL30568and HL34343 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/431,412, filed onMay 9, 2006, which is a continuation of U.S. Ser. No. 10/423,830, filedon Apr. 25, 2003, which is a continuation-in-part of 10/273,386, filedon Oct. 16, 2002, which is a continuation-in-part of 10/187,215, filedon Jun. 28, 2002, which is a continuation-in-part of 09/896,841, filedon Jun. 29, 2001, which is a continuation-in-part of U.S. Ser. No.09/645,454, filed on Aug. 24, 2000 all of which are incorporated hereinby reference in their entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to the field of atherosclerosis. In particular,this invention pertains to the identification of a class of peptidesthat are orally administrable and that ameliorate one or more symptomsof atherosclerosis.

BACKGROUND OF THE INVENTION

Cardiovascular disease is a leading cause of morbidity and mortality,particularly in the United States and in Western European countries.Several causative factors are implicated in the development ofcardiovascular disease including hereditary predisposition to thedisease, gender, lifestyle factors such as smoking and diet, age,hypertension, and hyperlipidemia, including hypercholesterolemia.Several of these factors, particularly hyperlipidemia andhypercholesteremia (high blood cholesterol concentrations) provide asignificant risk factor associated with atherosclerosis.

Cholesterol is present in the blood as free and esterified cholesterolwithin lipoprotein particles, commonly known as chylomicrons, very lowdensity lipoproteins (VLDLs), low density lipoproteins (LDLs), and highdensity lipoproteins (HDLs). Concentration of total cholesterol in theblood is influenced by (1) absorption of cholesterol from the digestivetract, (2) synthesis of cholesterol from dietary constituents such ascarbohydrates, proteins, fats and ethanol, and (3) removal ofcholesterol from blood by tissues, especially the liver, and subsequentconversion of the cholesterol to bile acids, steroid hormones, andbiliary cholesterol.

Maintenance of blood cholesterol concentrations is influenced by bothgenetic and environmental factors. Genetic factors include concentrationof rate-limiting enzymes in cholesterol biosynthesis, concentration ofreceptors for low density lipoproteins in the liver, concentration ofrate-limiting enzymes for conversion of cholesterols bile acids, ratesof synthesis and secretion of lipoproteins and gender of person.Environmental factors influencing the hemostasis of blood cholesterolconcentration in humans include dietary composition, incidence ofsmoking, physical activity, and use of a variety of pharmaceuticalagents. Dietary variables include amount and type of fat (saturated andpolyunsaturated fatty acids), amount of cholesterol, amount and type offiber, and perhaps amounts of vitamins such as vitamin C and D andminerals such as calcium.

Epidemiological studies show an inverse correlation of high densitylipoprotein (HDL) and apolipoprotein (apo) A-I levels with theoccurrence of atherosclerotic events (Wilson et al. (1988)Arteriosclerosis 8: 737-741). Injection of HDL into rabbits fed anatherogenic diet has been shown to inhibit atherosclerotic lesionformation (Badimon et al. (1990) J. Clin. Invest. 85: 1234-1241).

Human apo A-I has been a subject of intense study because of itsanti-atherogenic properties. Exchangeable apolipoproteins, including apoA-I, possess lipid-associating domains (Brouillette and Anantharamaiah(1995) Biochim. Biophys. Acta 1256:103-129; Segrest et al. (1974) FEBSLett. 38: 247-253). Apo A-I has been postulated to possess eight tandemrepeating 22mer sequences, most of which have the potential to formclass A amphipathic helical structures (Segrest et al. (1974) FEBS Lett.38: 247-253). Characteristics of the class A amphipathic helix includethe presence of positively charged residues at the polar-nonpolarinterface and negatively charged residues at the center of the polarface (Segrest et al. (1974) FEBS Lett. 38: 247-253; Segrest et al.(1990) Proteins: Structure, Function, and Genetics 8: 103-117). Apo A-Ihas been shown to strongly associate with phospholipids to formcomplexes and to promote cholesterol efflux from cholesterol-enrichedcells. The delivery and maintenance of serum levels of apo A-I toeffectively mitigate one or more symptoms of atherosclerosis hasheretofore proven elusive.

SUMMARY OF THE INVENTION

This invention provides novel peptides administration of which mitigatesone or more symptoms of atherosclerosis. In particular, it was adiscovery of this invention that peptides comprising a class Aamphipathic helix when formulated with “D” amino acid residue(s) and/orhaving protected amino and carboxyl termini can be orally administeredto an organism, are readily taken up and delivered to the serum, and areeffective to mitigate one or more symptoms of atherosclerosis. Incertain embodiments, the peptides can be formulated with all “L” aminoacid residues and are still effective, particular when administered byroutes other than oral administration.

The peptides of this invention are typically effective to stimulate theformation and cycling of pre-beta high density lipoprotein-likeparticles and/or to promote lipid transport and detoxification.

The peptides described herein are also effective for preventing theonset or inhibiting or eliminating one or more symptoms of osteoporosis.

It was also a surprising discovery that the peptides can be used toenhance (e.g. synergically enhance) the activity of statins therebypermitting the effective use of statins at lower dosages and/or causethe statins to be significantly more anti-inflammatory at any givendose.

Thus, in one embodiment, this invention provides a peptide thatameliorates one or more symptoms of atherosclerosis where the peptidecomprises a peptide or a concatamer of a peptide that ranges in lengthfrom about 10 to about 30 amino acids, that comprises at least one classA amphipathic helix; that protects a phospholipid against oxidation byan oxidizing agent; and that is not the D-18A peptide and/or is not apeptide disclosed in WO 97/36927, and/or U.S. Pat. No. 6,037,323, and/orU.S. Pat. No. 4,643,988. In certain embodiments, the peptide is at least10 amino acids in length. In certain embodiments, the peptide is about40 or fewer amino acids in length. In certain embodiments, the peptidecomprises all “L” amino acids while in certain other embodiments, thepeptide comprises at least one “D” amino acid residue. In certainembodiments, all enantiomeric amino acids comprising the peptide are “D”amino acids. The peptide can, optionally, further comprises a protectinggroup (e.g. a protecting group coupled to the amino and/or to thecarboxyl terminus). Suitable protecting groups include, but are notlimited to, acetyl (Ac), amide, a 3 to 20 carbon alkyl group, Fmoc,t-butoxycarbonyl (Tboc), 9-fluoreneacetyl group, 1-fluorenecarboxylicgroup, 9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt),4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl(MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl),Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom), cyclohexyloxy(cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), andtrifluoroacetyl (TFA). In certain embodiments, the peptide furthercomprises a first protecting group coupled to the amino terminus and asecond protecting group coupled to the carboxyl terminus. The peptidecan be mixed with a with a pharmacologically acceptable excipient (e.g.,a pharmacologically acceptable excipient suitable for oraladministration to a mammal). In certain embodiments, the comprises asequence selected from the group consisting ofD-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F- (SEQ ID NO: 2),D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F- (SEQ ID NO: 3),D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-(SEQ ID NO: 4),D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F- (SEQ ID NO: 5),D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F- (SEQ ID NO: 6),D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-(SEQ ID NO: 7),D-W-F-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F- (SEQ ID NO: 8),D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F- (SEQ ID NO: 9),D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-(SEQ ID NO: 10),D-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F- (SEQ ID NO: 11),D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F- (SEQ ID NO: 12),D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-(SEQ ID NO: 13),E-W-L-K-L-F-Y-E-K-V-L-E-K-F-K-E-A-F- (SEQ ID NO: 14),E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F- (SEQ ID NO: 15),E-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-(SEQ ID NO: 16),E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F- (SEQ ID NO: 17),E-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F- (SEQ ID NO: 18),E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-(SEQ ID NO: 19),E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F- (SEQ ID NO: 20),A-F-Y-D-K-V-A-E-K-L-K-E-A-F- (SEQ ID NO: 21),A-F-Y-D-K-V-A-E-K-F-K-E-A-F- (SEQ ID NO: 22),A-F-Y-D-K-V-A-E-K-F-K-E-A-F- (SEQ ID NO: 23),A-F-Y-D-K-F-F-E-K-F-K-E-F-F-(SEQ ID NO: 24),A-F-Y-D-K-F-F-E-K-F-K-E-F-F- (SEQ ID NO: 25),A-F-Y-D-K-V-A-E-K-F-K-E-A-F-(SEQ ID NO: 26),A-F-Y-D-K-V-A-E-K-L-K-E-F-F- (SEQ ID NO: 27),A-F-Y-D-K-V-F-E-K-F-K-E-A-F-(SEQ ID NO: 28),A-F-Y-D-K-V-F-E-K-L-K-E-F-F- (SEQ ID NO: 29),A-F-Y-D-K-V-A-E-K-F-K-E-F-F- (SEQ ID NO: 30),K-A-F-Y-D-K-V-F-E-K-F-K-E-F- (SEQ ID NO: 31),L-F-Y-E-K-V-L-E-K-F-K-E-A-F- (SEQ ID NO: 32),A-F-Y-D-K-V-A-E-K-F-K-E-A-F-(SEQ ID NO: 33),A-F-Y-D-K-V-A-E-K-L-K-E-F-F- (SEQ ID NO: 34),A-F-Y-D-K-V-F-E-K-F-K-E-A-F-(SEQ ID NO: 35),A-F-Y-D-K-V-F-E-K-L-K-E-F-F- (SEQ ID NO: 36),A-F-Y-D-K-V-A-E-K-F-K-E-F-F-(SEQ ID NO: 37),A-F-Y-D-K-V-F-E-K-F-K-E-F-F- (SEQ ID NO: 38),D-W-L-K-A-L-Y-D-K-V-A-E-K-L-K-E-A-L- (SEQ ID NO: 39),D-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-(SEQ ID NO: 40),D-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F- (SEQ ID NO: 41),E-W-L-K-A-L-Y-E-K-V-A-E-K-L-K-E-A-L- (SEQ ID NO: 42),E-W-L-K-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-(SEQ ID NO: 43),E-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F- (SEQ ID NO: 44),E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F- (SEQ ID NO: 45),E-W-L-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-(SEQ ID NO: 46),E-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F- (SEQ ID NO: 47),D-F-L-K-A-W-Y-D-K-V-A-E-K-L-K-E-A-W- (SEQ ID NO: 48),E-F-L-K-A-W-Y-E-K-V-A-E-K-L-K-E-A-W-(SEQ ID NO: 49),D-F-W-K-A-W-Y-D-K-V-A-E-K-L-K-E-W-W- (SEQ ID NO: 50),E-F-W-K-A-W-Y-E-K-V-A-E-K-L-K-E-W-W- (SEQ ID NO: 51),D-K-L-K-A-F-Y-D-K-V-F-E-W-A-K-E-A-F-(SEQ ID NO: 52),D-K-W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L-(SEQ ID NO: 53),E-K-L-K-A-F-Y-E-K-V-F-E-W-A-K-E-A-F- (SEQ ID NO: 54),E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L-(SEQ ID NO: 55),D-W-L-K-A-F-V-D-K-F-A-E-K-F-K-E-A-Y- (SEQ ID NO: 56),E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L- (SEQ ID NO: 57),D-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F-(SEQ ID NO: 58),E-W-L-K-A-F-V-Y-E-K-V-F-K-L-K-E-F-F-(SEQ ID NO: 59),D-W-L-R-A-F-Y-D-K-V-A-E-K-L-K-E-A-F- (SEQ ID NO: 60),E-W-L-R-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-(SEQ ID NO: 61),D-W-L-K-A-F-Y-D-R-V-A-E-K-L-K-E-A-F- (SEQ ID NO: 62),E-W-L-K-A-F-Y-E-R-V-A-E-K-L-K-E-A-F- (SEQ ID NO: 63),D-W-L-K-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-(SEQ ID NO: 64),E-W-L-K-A-F-Y-E-K-V-A-E-R-L-K-E-A-F- (SEQ ID NO: 65),D-W-L-K-A-F-Y-D-K-V-A-E-K-L-R-E-A-F- (SEQ ID NO: 66),E-W-L-K-A-F-Y-E-K-V-A-E-K-L-R-E-A-F-(SEQ ID NO: 67),D-W-L-K-A-F-Y-D-R-V-A-E-R-L-K-E-A-F- (SEQ ID NO: 68),E-W-L-K-A-F-Y-E-R-V-A-E-R-L-K-E-A-F- (SEQ ID NO: 69),D-W-L-R-A-F-Y-D-K-V-A-E-K-L-R-E-A-F-(SEQ ID NO: 70),E-W-L-R-A-F-Y-E-K-V-A-E-K-L-R-E-A-F- (SEQ ID NO: 71),D-W-L-R-A-F-Y-D-R-V-A-E-K-L-K-E-A-F- (SEQ ID NO: 72),E-W-L-R-A-F-Y-E-R-V-A-E-K-L-K-E-A-F-(SEQ ID NO: 73),D-W-L-K-A-F-Y-D-K-V-A-E-R-L-R-E-A-F- (SEQ ID NO: 74),E-W-L-K-A-F-Y-E-K-V-A-E-R-L-R-E-A-F- (SEQ ID NO: 75),D-W-L-R-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-(SEQ ID NO: 76),E-W-L-R-A-F-Y-E-K-V-A-E-R-L-K-E-A-F- (SEQ ID NO: 77),D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-P-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F(SEQ ID NO: 78),D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-P-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F(SEQ ID NO: 79),D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-P-D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F(SEQ ID NO: 80),D-K-L-K-A-F-Y-D-K-V-F-E-W-A-K-E-A-F-P-D-K-L-K-A-F-Y-D-K-V-F-E-W-L-K-E-A-F(SEQ ID NO: 81),D-K-W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L-P-D-K-W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L(SEQ ID NO: 82),D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-P-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F(SEQ ID NO: 83),D-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F-P-D-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F(SEQ ID NO: 84),D-W-L-K-A-F-Y-D-K-F-A-E-K-F-K-E-F-F-P-D-W-L-K-A-F-Y-D-K-F-A-E-K-F-K-E-F-F(SEQ ID NO: 85), E-W-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-A-F (SEQ ID NO: 86),D-W-F-K-A-F-Y-D-K-V-A-E-K-F (SEQ ID NO: 87), F-K-A-F-Y-D-K-V-A-E-K-F-K-E(SEQ ID NO: 88), F-K-A-F-Y-E-K-V-A-E-K-F-K-E (SEQ ID NO: 89),F-K-A-F-Y-D-K-V-A-E-K-F-K-E (SEQ ID NO: 90), F-K-A-F-Y-E-K-V-A-E-K-F-K-E(SEQ ID NO: 91), D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ ID NO: 92),E-W-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-A-F (SEQ ID NO: 93),A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ ID NO: 94), D-W-F-K-A-F-Y-D-K-V-A-E-K-F(SEQ ID NO: 95), D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F (SEQ ID NO: 96),E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F (SEQ ID NO: 97),A-F-Y-D-K-V-F-E-K-F-K-E-F-F (SEQ ID NO: 98), A-F-Y-E-K-V-F-E-K-F-K-E-F-F(SEQ ID NO: 99), D-W-L-K-A-F-Y-D-K-V-F-E-K-F (SEQ ID NO: 100),E-W-L-K-A-F-Y-E-K-V-F-E-K-F (SEQ ID NO: 101),L-K-A-F-Y-D-K-V-F-E-K-F-K-E (SEQ ID NO: 102), andL-K-A-F-Y-E-K-V-F-E-K-F-K-E (SEQ ID NO: 103). In certain embodiments,the foregoing peptides comprise all “L” amino acids. In certainembodiments, the foregoing peptides comprise at least one “D” aminoacid, more typically a plurality of “D” amino acids. In certainembodiments, at least half of the enantiomeric amino acids are “D” aminoacids, and in certain embodiments, all of the enantiomeric amino acidsare “D” amino acids.

In certain embodiments, the peptide further comprises a protecting groupcoupled to the amino and/or to the carboxyl terminus. Thus, for example,the peptide can comprise a protecting group coupled to the aminoterminal where the amino terminal protecting group is a protecting groupselected from the group consisting of a benzoyl group, an acetyl, apropionyl, a carbobenzoxy, a propyl, a butyl, a pentyl, a hexyl, anN-methyl anthranilyl, and a 3 to 20 carbon alkyl and/or the peptidecomprises a protecting group coupled to the carboxyl terminal where thecarboxyl terminal protecting group is an amide.

The oxidizing agent can be an agent such as hydrogen peroxide,13(S)-HPODE, 15(S)-HPETE, HPODE, HPETE, HODE, HETE, and the like. Incertain embodiments, the phospholipid is selected from the groupconsisting of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine(PAPC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine(SAPC)), 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine(SAPE).

The peptide can be provided as a pharmaceutical formulation, e.g.combined with a pharmaceutically acceptable excipient. The peptide canbe provided as a unit dosage formulation. In certain embodiments, the asa time release formulation (e.g. in a “time-release” matrix,microencapsulated, etc.).

In another embodiment, this invention provides a method of enhancing theactivity of a statin in a mammal. The method typically involvescoadministering with the statin an effective amount of one or more ofthe peptides described herein. In certain embodiments the statininclude, but is not limited to one or more statins selected from thegroup consisting of cerivastatin, atorvastatin, simvastatin,pravastatin, fluvastatin, lovastatin, rosuvastatin, and pitavastatin.The peptide can be administered before, simultaneously with, or afteradministration of the statin(s). In certain embodiments, the peptideand/or the statin are administered as a unit dosage formulation. Theadministration of the peptide and/or the statin can be by a routeincluding, but not limited to of oral administration, nasaladministration, rectal administration, intraperitoneal injection,intravascular injection, subcutaneous injection, transcutaneousadministration, intramuscular injection, and the like. In certainembodiments, the is a mammal diagnosed as having one or more symptoms ofatherosclerosis. In certain embodiments, the mammal is a mammaldiagnosed as at risk for stroke or atherosclerosis. The mammal can be ahuman or a non-human mammal.

In still another embodiment, this invention provides a method ofmitigating one or more symptoms associated with atherosclerosis in amammal. The method typically involves administering to the mammal aneffective amount of one or more statins (e.g., cerivastatin,atorvastatin, simvastatin, pravastatin, fluvastatin, lovastatin,rosuvastatin, pitavastatin, etc.); and an effective amount of one ormore peptides described herein, where the effective amount of the statinis lower than the effective amount of a statin administered without thepeptide. In certain embodiments, the effective amount of the peptide islower than the effective amount of the peptide administered without thestatin. The peptide(s) can be administered before, simultaneously with,or after the statin(s). The peptide and/or the statin can beadministered as a unit dosage formulation. The peptide(s) and/orstatin(s) can be administered by a route including, but not limited tooral administration, nasal administration, rectal administration,intraperitoneal injection, intravascular injection, subcutaneousinjection, transcutaneous administration, inhalation administration, andintramuscular injection. In certain embodiments, the mammal is a humanor non-human mammal diagnosed as having one or more symptoms ofatherosclerosis and/or at risk for stroke and/or atherosclerosis.

This invention also provides a pharmaceutical formulation. Theformulation typically comprises a pharmaceutically acceptable excipientand one or more of the peptides described herein. Another pharmaceuticalformulation typically comprises one or more statins (e.g., cerivastatin,atorvastatin, simvastatin, pravastatin, fluvastatin, lovastatin,rosuvastatin, pitavastatin, etc.) and one or more peptides as describedherein. In certain embodiments, the peptide and/or said statin arepresent in an effective dose. In certain embodiments, the effectiveamount of the statin is lower than the effective amount of the statinadministered without the peptide(s) and/or the effective amount of thepeptide(s) is lower than the effective amount of the peptide(s)administered without the statin(s). In various embodiments, thestatin(s) and/or the peptide(s) are in a time release formulation (e.g.a time release matrix, a microencapsulated formulation, and the like).The pharmaceutical formulation can be a unit dosage formulation, e.g.,for oral administration. In certain embodiments, the formulation isformulated for administration by a route selected from the groupconsisting of oral administration, nasal administration, rectaladministration, intraperitoneal injection, intravascular injection,subcutaneous injection, transcutaneous administration, inhalationadministration, and intramuscular injection. The formulation can,optionally, further comprises one or more phospholipids (e.g. aphospholipid as described in U.S. Ser. No. 09/994,227).

In another embodiment, this invention provides a method of reducing orinhibiting one or more symptoms of osteoporosis in a mammal. The methodtypically involves administering to the mammal one or more peptides asdescribed herein, where the peptide is administered in a concentrationsufficient to reduce or eliminate one or more symptoms of osteoporosis.In certain embodiments, the peptide is administered in a concentrationsufficient to reduce or eliminate decalcification of a bone. In certainembodiments, the peptide is administered in a concentration sufficientto induce recalcification of a bone. The peptide(s) can be mixed with apharmacologically acceptable excipient, e.g. as described herein.

In certain embodiments, the methods and/or peptides of this inventionexclude any one or more peptides disclosed in WO 97/36927, and/or U.S.Pat. No. 6,037,323, and/or U.S. Pat. No. 4,643,988 and/or in Garber etal. (1992) Arteriosclerosis and Thrombosis, 12: 886-894. In certainembodiments this invention excludes any one or more peptides disclosedin U.S. Pat. No. 4,643,988 and/or in Garber et al (1992) that weresynthesized with all enantiomeric amino acids being L amino acids orsynthesized with D amino acids where the peptides possess blockinggroups. In certain embodiments, this invention excludes peptides havingthe formula A₁-B₁-B₂-C₁-D-B₃-B₄-A₂-C₂-B₅-B₆-A₃-C₃-B₇-C₄-A₄-B₈-B₉ (SEQ IDNO: 104) wherein A₁, A₂, A₃ and A₄ are independently aspartic acid orglutamic acid, or homologues or analogues thereof; B₁, B₂, B₃, B₄, B₅,B₆, B₇, B₈ and B₉ are independently tryptophan, phenylalanine, alanine,leucine, tyrosine, isoleucine, valine or α-naphthylalanine, orhomologues or analogues thereof; C₁, C₂, C₃ and C₄ are independentlylysine or arginine, and D is serine, threonine, alanine, glycine,histidine, or homologues or analogues thereof; provided that, when A₁and A₂ are aspartic acid, A₃ and A₄ are glutamic acid, B₂ and B₉ areleucine, B₃ and B₇ are phenylalanine, B₄ is tyrosine, B₅ is valine, B₆,B₈, and D are alanine, and C₁, C₂, C₃ and C₄ are lysine, B₁ is nottryptophan.

In certain embodiments, this invention excludes any one or more peptidesin WO 97/36927 and/or D variants thereof. Particular embodiments excludeone or more of the following: apoprotein A, apoprotein A-1, apoproteinA-2, apoprotein A4, apoprotein B, apoprotein B-48, apoprotein B-100,apoprotein C, apoprotein C-1, apoprotein C-2, apoprotein C-3, apoproteinD, apoprotein E as described in WO 97/36927.

In certain embodiments, also excluded are any one or more peptidesdisclosed in U.S. Pat. No. 6,037,323 and/or D variants thereof.Particular embodiments exclude apo A-I agonist compounds comprising (i)an 18 to 22-residue peptide or peptide analogue that forms anamphipathic .alpha.-helix in the presence of lipids and that comprisesthe formula:Z₁-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-Z₂(SEQ ID NO: 105) where X₁ is Pro (P), Ala (A), Gly (G), Asn (N), Gln (Q)or D-Pro (p); X₂ is an aliphatic amino acid; X₃ is Leu (L); X₄ is anacidic amino acid; X₅ is Leu (L) or Phe (F); X₆ is Leu (L) or Phe (F);X₇ is a basic amino acid; X₈ is an acidic amino acid; X₉ is Leu (L) orTrp (W); X₁₀ is Leu (L) or Trp (W); X₁₁ is an acidic amino acid or Asn(N); X₁₂ is an acidic amino acid; X₁₃ is Leu (L), Trp (W) or Phe (F);X₁₄ is a basic amino acid or Leu (L); X₁₅ is Gln (Q) or Asn (N); X₁₆ isa basic amino acid; X₁₇ is Leu (L); X₁₈ is a basic amino acid; Z₁ isH₂N—or RC(O)NH—; Z₂ is —C(O)NRR, —C(O)OR or —C(O)OH or a salt thereof;each R is independently —H, (C₁-C₆) alkyl, (C₁-C₆) alkenyl, (C₁-C₆)alkynyl, (C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl or6-26 membered alkheteroaryl or a 1 to 4-residue peptide or peptideanalogue in which one or more bonds between residues 1-7 areindependently a substituted amide, an isostere of an amide or an amidemimetic; and each “-” between residues X₁ through X₁₈ independentlydesignates an amide linkage, a substituted amide linkage, an isostere ofan amide or an amide mimetic; or (ii) an altered form of formula (I) inwhich at least one of residues X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀,X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇ or X₁₈ is conservatively substitutedwith another residue, and/or D variants thereof.

DEFINITIONS

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “class A amphipathic helix” refers to a protein structure thatforms an α-helix producing a segregation of a polar and nonpolar faceswith the positively charged residues residing at the polar-nonpolarinterface and the negatively charged residues residing at the center ofthe polar face (see, e.g., “Segrest et al. (1990) Proteins: Structure,Function, and Genetics 8: 103-117).

The term “ameliorating” when used with respect to “ameliorating one ormore symptoms of atherosclerosis” refers to a reduction, prevention, orelimination of one or more symptoms characteristic of atherosclerosisand/or associated pathologies. Such a reduction includes, but is notlimited to a reduction or elimination of oxidized phospholipids, areduction in atherosclerotic plaque formation and rupture, a reductionin clinical events such as heart attack, angina, or stroke, a decreasein hypertension, a decrease in inflammatory protein biosynthesis,reduction in plasma cholesterol, and the like. “Ameliorating one or moresymptoms of atherosclerosis” can also refer to improving blood flow tovascular beds affected by atherosclerosis.

The term “enantiomeric amino acids” refers to amino acids that can existin at least two forms that are nonsuperimposable mirror images of eachother. Most amino acids (except glycine) are enantiomeric and exist in aso-called L-form (L amino acid) or D-form (D amino acid). Most naturallyoccurring amino acids are “L” amino acids. The terms “D amino acid” and“L amino acid” are used to refer to absolute configuration of the aminoacid, rather than a particular direction of rotation of plane-polarizedlight. The usage herein is consistent with standard usage by those ofskill in the art.

The term “protecting group” refers to a chemical group that, whenattached to a functional group in an amino acid (e.g. a side chain, analpha amino group, an alpha carboxyl group, etc.) blocks or masks theproperties of that functional group. Preferred amino-terminal protectinggroups include, but are not limited to acetyl, or amino groups. Otheramino-terminal protecting groups include, but are not limited to alkylchains as in fatty acids, propionyl, formyl and others. Preferredcarboxyl terminal protecting groups include, but are not limited togroups that form amides or esters.

The phrase “protect a phospholipid from oxidation by an oxidizing agent”refers to the ability of a compound to reduce the rate of oxidation of aphospholipid (or the amount of oxidized phospholipid produced) when thatphospholipid is contacted with an oxidizing agent (e.g. hydrogenperoxide, 13-(S)-HPODE, 15-(S)-HPETE, HPODE, HPETE, HODE, HETE, etc.).

The terms “low density lipoprotein” or “LDL” is defined in accordancewith common usage of those of skill in the art. Generally, LDL refers tothe lipid-protein complex which when isolated by ultracentrifugation isfound in the density range d=1.019 to d=1.063.

The terms “high density lipoprotein” or “HDL” is defined in accordancewith common usage of those of skill in the art. Generally “HDL” refersto a lipid-protein complex which when isolated by ultracentrifugation isfound in the density range of d=1.063 to d=1.21.

The term “Group I HDL” refers to a high density lipoprotein orcomponents thereof (e.g. apo A-I, paraoxonase, platelet activatingfactor acetylhydrolase, etc.) that reduce oxidized lipids (e.g. in lowdensity lipoproteins) or that protect oxidized lipids from oxidation byoxidizing agents.

The term “Group II HDL” refers to an HDL that offers reduced activity orno activity in protecting lipids from oxidation or in repairing (e.g.reducing) oxidized lipids.

The term “HDL component” refers to a component (e.g. molecules) thatcomprises a high density lipoprotein (HDL). Assays for HDL that protectlipids from oxidation or that repair (e.g. reduce oxidized lipids) alsoinclude assays for components of HDL (e.g. apo A-I, paraoxonase,platelet activating factor acetylhydrolase, etc.) that display suchactivity.

The term “human apo A-I peptide” refers to a full-length human apo A-Ipeptide or to a fragment or domain thereof comprising a class Aamphipathic helix.

A “monocytic reaction” as used herein refers to monocyte activitycharacteristic of the “inflammatory response” associated withatherosclerotic plaque formation. The monocytic reaction ischaracterized by monocyte adhesion to cells of the vascular wall (e.g.cells of the vascular endothelium), and/or chemotaxis into thesubendothelial space, and/or differentiation of monocytes intomacrophages.

The term “absence of change” when referring to the amount of oxidizedphospholipid refers to the lack of a detectable change, more preferablythe lack of a statistically significant change (e.g. at least at the85%, preferably at least at the 90%, more preferably at least at the95%, and most preferably at least at the 98% or 99% confidence level).The absence of a detectable change can also refer to assays in whichoxidized phospholipid level changes, but not as much as in the absenceof the protein(s) described herein or with reference to other positiveor negative controls.

The following abbreviations are used herein: PAPC:L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC:1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine; PGPC:1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC:1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phosphocholine; ChC18:2: cholesteryl linoleate; ChC18:2-OOH: cholesteryl linoleatehydroperoxide; DMPC: 1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine;PON: paraoxonase; HPF: Standardized high power field; PAPC:L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC:1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine; PGPC:1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC:1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phosphocholine;PON: paraoxonase; BL/6: C57BL/6J; C3H:C3H/HeJ.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity (e.g. for lipoproteins)) or bindingaffinity (e.g. for lipids or lipoproteins)) of the molecule. Typicallyconservative amino acid substitutions involve substitution one aminoacid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.With respect to the peptides of this invention sequence identity isdetermined over the full length of the peptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins & Sharp (1989) CABIOS 5: 151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov).This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad.Sci. USA, 90: 5873-5787). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

The term “D-18A peptide” refers to a peptide having the sequence:D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F (SEQ ID NO: 1) where all of theenantiomeric amino acids are D form amino acids.

The term “coadministering” or “concurrent administration”, when used,for example with respect to a peptide of this invention and anotheractive agent (e.g. a statin), refers to administration of the peptideand the active agent such that both can simultaneously achieve aphysiological effect. The two agents, however, need not be administeredtogether. In certain embodiments, administration of one agent canprecede administration of the other, however, such coadministeringtypically results in both agents being simultaneously present in thebody (e.g. in the plasma) at a significant fraction (e.g. 20% orgreater, preferably 30% or 40% or greater, more preferably 50% or 60% orgreater, most preferably 70% or 80% or 90% or greater) of their maximumserum concentration for any given dose.

The term “detoxify” when used with respect to lipids, LDL, or HDL refersthe removal of some or all oxidizing lipids and/or oxidized lipids.Thus, for example, the uptake of all or some HPODE and/or HPETE (bothhydroperoxides on fatty acids) will prevent or reduce entrance of theseperoxides into LDLs and thus prevent or reduce LDL oxidation.

The term “pre-beta high density lipoprotein-like particles” typicallyrefers to cholesterol containing particles that also contain apoA-I andwhich are smaller and relatively lipid-poor compared to thelipid:protein ratio in the majority of HDL particles. When plasma isseparated by FPLC, these “pre-beta high density lipoprotein-likeparticles” are found in the FPLC fractions containing particles smallerthan those in the main HDL peak and are located to the right of HDL inan FPLC chromatogram as shown in the accompanying FIGS. 5 and 8.

The phrase “reverse lipid transport and detoxification” refers to theremoval of lipids including cholesterol, other sterols includingoxidized sterols, phospholipids, oxidizing agents, and oxidizedphospholipids from tissues such as arteries and transport out of theseperipheral tissues to organs where they can be detoxified and excretedsuch as excretion by the liver into bile and excretion by the kidneysinto urine. Detoxification also refers to preventing the formationand/or destroying oxidized phospholipids as explained herein.

The term “biological sample” as used herein refers to any sampleobtained from a living organism or from an organism that has died.Examples of biological samples include body fluids, tissue specimens,cells and cell lines taken from an organism (e.g. a human or non-humanmammal).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the detection of ¹⁴C-D-4F in mouse plasma. 22 μg of¹⁴C-D-4F, 140,000 DPM in 100 μl of water was administered to 4 month oldfemale apoE null mice by stomach tube. Blood samples were obtained ateach time point (n=4 mice per time point) and plasma ¹⁴C-radioactivitydetermined in 1.0 ml of plasma

FIG. 2 illustrates the blood concentration of D-4F. Twenty-two μg of¹⁴C-D-4F, 140,000 DPM in 100 μl of water was administered to 4-month-oldfemale apoE null mice via tail vein injection (n=4 mice for each timepoint). Blood samples were obtained at the indicated time points and¹⁴C-radioactivity in 1.0 ml of plasma was determined.

FIG. 3 shows the results from a Western blot for mouse apoA-I. ApoE nullmice were given 500 μg D-4F (+D-4F) or not given (no D-4F) by stomachtube 20 min prior to being bled. Plasma was separated by FPLC andfractions 30, 35, 36, and 37 were analyzed by native-PAGE and Westernblotting using antisera to mouse apoA-I. The diameter of the particlesis shown on the left. (FPLC Fxn=FPLC fraction number; HDL Peak=fraction30; CCP=fractions 35 to 37).

FIGS. 4A, 4B, and 4C show the results of an LC-MRM analysis. Twenty minafter instilling 500 μg D-4F into the stomachs of apoE null mice, themice were bled and their plasma was fractionated by FPLC and thefractions analyzed by LC-MRM after adding D-5F as an internal standard.FIG. 4A shows a solvent blank, FIG. 4B shows standards for D-4F (top)and D-5F (bottom), and FIG. 4C shows the results of analysis of pooledFPLC fractions 35-37 for D-4F in the top panel with the internalstandard D-5F in the bottom panel.

FIG. 5 illustrates cholesterol and paraoxonase Activity. Twenty minafter instilling saline (No D-4F, top panel) or 500 μg D-4F (+D-4F,bottom panel) into the stomachs of apoE null mice, the mice were bledand their plasma fractionated by FPLC and cholesterol (thin black line)and paraoxonase (PON) activity (thick black line) determined in thefractions. The bottom panel shows the fractions with cholesterolcontaining particles (CCP) that appeared to the right of HDL after D-4F.As shown in the bottom panel, the CCP fractions also contained PONactivity after D-4F.

FIG. 6 illustrates the ability of the pre-beta HDL-like particles (CCP)in FIG. 5 to detoxify lipids and prevent human artery wall cells fromproducing an inflammatory reaction (monocyte chemotactic activity).Water or 500 □g of D-4F were instilled into the stomachs of apoE nullmice. Twenty minutes later the mice were bled and their plasmafractionated by FPLC. Fractions 28-30 contained HDL and fractions 35-37contained the pre-beta HDL-like particles (CCP). Twenty μg of1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) wasadded together with 1 μg/ml of hydroperoxyeicosatetraenoic acid (HPODE)to cocultures of human artery wall cells as described previously (Navabet al. (2001) J Lipid Res., 42: 1308-1317). Human HDL (h, HDL) was addedat 350 μg/ml cholesterol or no addition was made to the cocultures (NoAddition), or mouse HDL isolated by FPLC (fractions 28-30) from the micegiven water alone (Water Control HDL) or D-4F (D-4F HDL) at 50 μg/mlHDL-cholesterol or the pre-beta HDL-like particle fractions (fractions35-37) after water alone (Water Control CCP) or after D-4F (D-4F CCP) at10 μg/ml cholesterol were added to the cocultures. After 8 hours ofincubation, supernatants were collected and assayed for monocytechemotactic activity using standard neuroprobe chambers. The data aremean±SD of the number of migrated monocytes in 9 fields, for triplicatesamples.

FIG. 7 illustrates the formula of N-methyl anthranilyl-D-4F. Only twoterminal amino acids of the 4F polypeptide are shown.

FIGS. 8A, 8B, and 8C illustrate the time course of D4F and plasmacholesterol. LDL receptor null female mice at 8 weeks of age (5 mice pergroup) were given by stomach tube 22 μg/mouse of N-MethylAnthranilyl-D-4F and were then bleed 1, 2, or 8 hours later. Theirplasma was fractionated by FPLC and analyzed for cholesterol andfluorescence. The 1-hour time point is shown in FIG. 8A, the 2 hour timepoint is shown in FIG. 8B, and the 8 hour time point is shown in FIG.8C.

FIG. 9 illustrates the synergy between statins and D-4F in restoring HDLprotective capacity. ApoE null female mice three months old on a chowdiet were given drinking water alone (Water), or drinking watercontaining 1 μg/ml of D-4F, or 0.05 mg/ml of Atorvastatin, or 0.05 mg/mlof Pravastatin, or 1 μg/ml of D-4F together with 0.05 mg/ml ofAtorvastatin, or 1 μg/ml of D-4F together with 0.05 mg/ml ofPravastatin. After 24 hours the mice were bled and their HDL was testedin a human artery wall coculture model. Twenty μg of1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) wasadded together with 1 μg/ml of hydroperoxyeicosatetraenoic acid (HPODE)to cocultures of human artery wall cells as described previously (Navabet al. (2001) J Lipid Res., 42: 1308-1317). Human HDL (h, HDL) was addedat 350 μg/ml cholesterol or no addition was made to the cocultures (NoAddition), or mouse HDL isolated by FPLC from the mice given drinkingwater alone (Water) or the additions shown on the X-axis were added tothe cocultures at 50 μg/ml HDL-cholesterol. After 8 hours of incubation,supernatants were collected and assayed for monocyte chemotacticactivity using standard neuroprobe chambers. The data are mean±SD of thenumber of migrated monocytes in 9 fields, for triplicate samples fromeach of two separate experiments. Asterisks indicate statisticallysignificant difference at the level of p<0.05 between the values for theindividual compounds as compared to those for the combination.

DETAILED DESCRIPTION

This invention pertains to the discovery that synthetic peptidesdesigned to mimic the class A amphipathic helical motif (Segrest et al.(1990) Proteins: Structure, Function, and Genetics 8: 103-117) are ableto associate with phospholipids and exhibit many biological propertiessimilar to human apo-A-I. In particular, it was a discovery of thisinvention that when such peptides are formulated using D amino acids,the peptides show dramatically elevated serum half-lives and,particularly when the amino and/or carboxy termini are blocked, can evenbe orally administered.

It was also a surprising discovery that these peptides can stimulate theformation and cycling of pre-beta high density lipoprotein-likeparticles. In addition, the peptides are capable ofenhancing/synergizing the effect of statins allowing statins to beadministered as significantly lower dosages or to be significantly moreanti-inflammatory at any given dose. It was also discovered that thepeptides described herein can inhibit and/or prevent and/or treat one ormore symptoms of osteoporosis.

Moreover, it was a surprising discovery of this invention that suchD-form peptides retain the biological activity of the correspondingL-form peptide. In vivo animal studies using such D-form peptides showedeffective oral delivery, elevated serum half-life, and the ability tomitigate or prevent/inhibit one or more symptoms of atherosclerosis.

I. Stimulating the Formation and Cycling of Pre-Beta High DensityLipoprotein-Like Particles.

Reverse cholesterol transport is considered to be important inpreventing the build up of lipids that predisposes to atherosclerosis(Shah et al. (2001) Circulation, 103: 3047-3050.) Many have believed thelipid of consequence is cholesterol. Our laboratory has shown that thekey lipids are oxidized phospholipids that initiate the inflammatoryresponse in atherosclerosis (Navab et al. (2001) Arterioscler ThrombVasc Biol., 21(4): 481-488; Van Lenten et al. (001) Trends CardiovascMed, 11: 155-161; Navab M et al. (2001) Circulation, 104: 2386-2387).

This inflammatory response is also likely responsible for plaque erosionor rupture that leads to heart attack and stroke. HDL-cholesterol levelsare inversely correlated with risk for heart attack and stroke (Downs etal. (1998) JAMA 279: 1615-1622; Gordon et al. (1977) Am J Med., 62:707-714; Castelli et al. (1986) JAMA, 256: 2835-2838).

Pre-beta HDL is generally considered to be the most active HDL fractionin promoting reverse cholesterol transport (e.g., picking up cholesterolfrom peripheral tissues such as arteries and carrying it to the liverfor excretion into the bile; see, Fielding and Fielding (2001) BiochimBiophys Acta, 1533(3): 175-189). However, levels of pre-beta HDL can beincreased because of a failure of the pre-beta HDL to be cycled intomature alpha-migrating HDL e.g. LCAT deficiency or inhibition (O'Connoret al. (1998) J Lipid Res, 39: 670-678). High levels of pre-beta HDLhave been reported in coronary artery disease patients (Miida et al.(1996) Clin Chem., 42: 1992-1995).

Moreover, men have been found to have higher levels of pre-beta HDL thanwomen but the risk of men for coronary heart disease is greater than forwomen (O'Connor et al. (1998) J Lipid Res., 39: 670-678). Thus, staticmeasurements of pre-beta HDL levels themselves are not necessarilypredictive of risk for coronary artery disease. The cycling, however, ofcholesterol through pre-beta HDL into mature HDL is universallyconsidered to be protective against atherosclerosis (Fielding andFielding (2001) Biochim Biophys Acta, 1533(3): 175-189). Moreover, wehave demonstrated that the removal of oxidized lipids from artery wallcells through this pathway protects against LDL oxidation.

As described herein in Example 1, despite relatively low absorptionrates when orally administered, the peptides of this invention (e.g.D-4F) were highly active.

In studies of Apo-E null mice orally administered D-4F, we determinedthat 20 min after absorption from the intestine, D-4F forms smallpre-beta HDL-like particles that contain relatively high amounts ofapoA-I and paraoxonase. Indeed, estimating the amount of apoA-I in thesepre-beta HDL-like particles from Western blots and comparing the amountof apoA-I to the amount of D-4F in these particles (determined byradioactivity or LC-MRM) suggests that as D-4F is absorbed from theintestine, it acts as a catalyst causing the formation of these pre-betaHDL-like particles. This small amount of intestinally derived D-4Fappears to recruit amounts of apoA-I, paraoxonase, and cholesterol intothese particles that are orders of magnitude more than the amount ofD-4F.

Thus, following absorption, D-4F rapidly recruits relatively largeamounts of apoA-I and paraoxonase to form pre-beta HDL-like particleswhich are very likely the most potent particles for both promotingreverse cholesterol transport and for destroying biologically activeoxidized lipids. We believe that the formation of these particles andtheir subsequent rapid incorporation into mature HDL likely explains thedramatic reduction in atherosclerosis that we observed in LDL receptornull mice on a Western diet and in apoE-null mice on a chow dietindependent of changes in plasma cholesterol or HDL-cholesterol.

Thus, in one embodiment, this invention provides methods of stimulatingthe formation and cycling of pre-beta high density lipoprotein-likeparticles by administration of one or more peptides as described herein.The peptides can thereby promote lipid transport and detoxification.

II. Synergizing the Activity of Statins.

As demonstrated in Example 2, adding a low dosage of D-4F (1 μg/ml) tothe drinking water of apoE null mice for 24 hours did not significantlyimprove HDL function (see, e.g., FIG. 9). FIG. 9 also shows that adding0.05 mg/ml of atorvastatin or pravastatin alone to the drinking water ofthe apoE null mice for 24 hours did not improve HDL function. However,when D-4F 1 μg/ml was added to the drinking water together with 0.05mg/ml of atorvastatin or pravastatin there was a significant improvementin HDL function (see, e.g., FIG. 9). Indeed the pro-inflammatory apoEnull HDL became as anti-inflammatory as 350 μg/ml of normal human HDL(h, HDL).

Thus, doses of D-4F alone, or statins alone, which by themselves had noeffect on HDL function when given together acted synergistically. WhenD-4F and a statin were given together to apo E null mice, theirpro-inflammatory HDL at 50 μg/ml of HDL-cholesterol became as effectiveas normal human HDL at 350 μg/ml of HDL-cholesterol in preventing theinflammatory response induced by the action of HPODE oxidizing PAPC incocultures of human artery wall cells.

Thus, in certain embodiments this invention provides methods forenhancing the activity of statins. The methods generally involveadministering one or more peptides as described herein concurrently withone or more statins. The D-4F or other similar peptides as describedherein achieve synergistic action between the statin and the orallypeptide(s) to ameliorate atherosclerosis. In this context statins can beadministered at significantly lower dosages thereby avoiding variousharmful side effects (e.g. muscle wasting) associated with high dosagestatin use and/or the anti-inflammatory properties of statins at anygiven dose are significantly enhanced.

III. Inhibiting/Treating Osteoporosis.

Vascular calcification and osteoporosis often co-exist in the samesubjects (Ouchi et al. (1993) Ann NY Acad Sci., 676: 297-307; Boukhrisand Becker (1972) JAMA, 219: 1307-1311; Banks et al. (1994) Eur J ClinInvest., 24: 813-817; Laroche et al. (1994) Clin Rheumatol., 13:611-614; Broulik and Kapitola (1993) Endocr Regul., 27: 57-60; Frye etal. (1992) Bone Mine., 19: 185-194; Barengolts et al. (1998) CalcifTissue Int., 62: 209-213; Burnett and Vasikaran (2002) Ann ClinBiochem., 39: 203-210. Parhami et al. (Parhami et al. (1997) ArteriosclThromb Vasc Biol., 17: 680-687) demonstrated that mildly oxidized LDL(MM-LDL) and the biologically active lipids in MM-LDL [i.e. oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine) (Ox-PAPC)],as well as the isoprostane, 8-iso prostaglandin E₂, but not theunoxidized phospholipid (PAPC) or isoprostane 8-iso progstaglandinF_(2α) induced alkaline phosphatase activity and osteoblasticdifferentiation of calcifying vascular cells (CVCs) in vitro, butinhibited the differentiation of MC3T3-E1 bone cells.

The osteon resembles the artery wall in that the osteon is centered onan endothelial cell-lined lumen surrounded by a subendothelial spacecontaining matrix and fibroblast-like cells, which is in turn surroundedby preosteoblasts and osteoblasts occupying a position analogous tosmooth muscle cells in the artery wall (Id.). Trabecular boneosteoblasts also interface with bone marrow subendothelial spaces (Id.).Parhami et al. postulated that lipoproteins could cross the endotheliumof bone arteries and be deposited in the subendothelial space where theycould undergo oxidation as in coronary arteries (Id.). Based on their invitro data they predicted that LDL oxidation in the subendothelial spaceof bone arteries and in bone marrow would lead to reduced osteoblasticdifferentiation and mineralization which would contribute toosteoporosis (Id.). Their hypothesis further predicted that LDL levelswould be positively correlated with osteoporosis as they are withcoronary calcification (Pohle et al. (2001) Circulation, 104: 1927-1932)but HDL levels would be negatively correlated with osteoporosis (Parhamiet al. (1997) Arterioscl Thromb Vasc Biol., 17: 680-687).

In vitro, the osteoblastic differentiation of the marrow stromal cellline M2-10B4 was inhibited by MM-LDL but not native LDL (Parhami et al.(1999) J Bone Miner Res., 14: 2067-2078). When marrow stromal cells fromatherosclerosis susceptible C57BL/6 (BL6) mice fed a low fat chow dietwere cultured there was robust osteogenic differentiation (Id.). Incontrast, when the marrow stromal cells taken from the mice after a highfat, atherogenic diet were cultured they did not undergo osteogenicdifferentiation (Id.). This observation is particularly important sinceit provides a possible explanation for the decreased osteogenicpotential of marrow stromal cells in the development of osteoporosis(Nuttall and Gimble (2000) Bone, 27: 177-184). In vivo the decrease inosteogenic potential is accompanied by an increase in adipogenesis inosteoporotic bone (Id.).

It was found that adding D-4F to the drinking water of apoE null micefor 6 weeks dramatically increased trabecular bone mineral density(Example 3).

The data indicate that osteoporosis can be regarded as an“atherosclerosis of bone”. It appears to be a result of the action ofoxidized lipids. HDL destroys these oxidized lipids and promotesosteoblastic differentiation. The data illustrated in Example 3 indicatethat administering peptide(s) of this invention to a mammal (e.g. in thedrinking water of apoE null mice) dramatically increases trabecular bonein just a matter of weeks.

This indicates that the peptides described herein are useful formitigation one or more symptoms of atherosclerosis (e.g. for inhibitingdecalcification) or for inducing recalcification of osteoporotic bone.The peptides are also useful as prophylactics to prevent the onset ofsymptom(s) of osteoporosis in a mammal (e.g. a patient at risk forosteoporosis).

IV. Mitigation of a Symptom of Atherosclerosis.

We discovered that normal HDL inhibits three steps in the formation ofmildly oxidized LDL. In those studies (see, copending application U.S.Ser. No. 09/541,468, filed on Mar. 31, 2000) we demonstrated thattreating human LDL in vitro with apo A-I or an apo A-I mimetic peptide(37 pA) removed seeding molecules from the LDL that included HPODE andHPETE. These seeding molecules were required for cocultures of humanartery wall cells to be able to oxidize LDL and for the LDL to inducethe artery wall cells to produce monocyte chemotactic activity. We alsodemonstrated that after injection of apo A-I into mice or infusion intohumans, the LDL isolated from the mice or human volunteers afterinjection/infusion of apo A-I was resistant to oxidation by human arterywall cells and did not induce monocyte chemotactic activity in theartery wall cell cocultures.

The protective function of the D peptides of this invention isillustrated in the parent applications (Ser. No. 09/645,454, filed Aug.24, 2000, Ser. No. 09/896,841, filed Jun. 29, 2001, and WO 02/15923(PCT/US01/26497), filed Jun. 29, 2001, see, e.g., FIGS. 1-5 in WO02/15923. FIG. 1, panels A, B, C, and D in WO 02/15923 show theassociation of ¹⁴C-D-5F with blood components in an ApoE null mouse. Itis also demonstrated that HDL from mice that were fed an atherogenicdiet and injected with PBS failed to inhibit the oxidation of human LDLand failed to inhibit LDL-induced monocyte chemotactic activity in humanartery wall cocultures. In contrast, HDL from mice fed an atherogenicdiet and injected daily with peptides described herein was as effectivein inhibiting human LDL oxidation and preventing LDL-induced monocytechemotactic activity in the cocultures as was normal human HDL (FIGS. 2Aand 2B in WO 02/15923). In addition, LDL taken from mice fed theatherogenic diet and injected daily with PBS was more readily oxidizedand more readily induced monocyte chemotactic activity than LDL takenfrom mice fed the same diet but injected with 20 μg daily of peptide 5F.The D peptide did not appear to be immunogenic (FIG. 4 in WO 02/15923).

The in vitro responses of human artery wall cells to HDL and LDL frommice fed the atherogenic diet and injected with a peptide according tothis invention are consistent with the protective action shown by suchpeptides in vivo. Despite, similar levels of total cholesterol,LDL-cholesterol, IDL+VLDL-cholesterol, and lower HDL-cholesterol as apercent of total cholesterol, the animals fed the atherogenic diet andinjected with the peptide had significantly lower lesion scores (FIG. 5in WO 02/15923). The peptides of this invention thus preventedprogression of atherosclerotic lesions in mice fed an atherogenic diet.

Thus, in one embodiment, this invention provides methods forameliorating and/or preventing one or more symptoms of atherosclerosis.

VI. Mitigation of a Symptom of Atheroscloerosis Associated with an AcuteInflammatory Response.

The peptides of this invention are also useful in a number of contexts.For example, we have observed that cardiovascular complications (e.g.atherosclerosis, stroke, etc.) frequently accompany or follow the onsetof an acute phase inflammatory response. Such an acute stateinflammatory response is often associated with a recurrent inflammatorydisease (e.g., leprosy, tuberculosis, systemic lupus erythematosus, andrheumatoid arthritis), a viral infection (e.g. influenza), a bacterialinfection, a fungal infection, an organ transplant, a wound or othertrauma, an implanted prosthesis, a biofilm, and the like.

It was a surprising discovery of this invention that administration ofone or more of the peptides described herein, can reduce or prevent theformation of oxidized phospholipids during or following an acute phaseresponse and thereby mitigate or eliminate cardiovascular complicationsassociated with such a condition.

Thus, for example, we have demonstrated that a consequence of influenzainfection is the diminution in paraoxonase and platelet activatingacetylhydrolase activity in the HDL. Without being bound by a particulartheory, we believe that, as a result of the loss of these HDL enzymaticactivities and also as a result of the association of pro-oxidantproteins with HDL during the acute phase response, HDL is no longer ableto prevent LDL oxidation and was no longer able to prevent theLDL-induced production of monocyte chemotactic activity by endothelialcells.

We observed that in a subject injected with very low dosages of thepolypeptides of this invention (e.g. 20 micrograms for mice) daily afterinfection with the influenza A virus paraoxonase levels did not fall andthe biologically active oxidized phospholipids were not generated beyondbackground. This indicates that D-4F (and/or other peptides of thisinvention) can be administered (e.g. orally or by injection) to patientswith known coronary artery disease during influenza infection or otherevents that can generate an acute phase inflammatory response (e.g. dueto viral infection, bacterial infection, trauma, transplant, variousautoimmune conditions, etc.) and thus we can prevent by this short termtreatment the increased incidence of heart attack and stroke associatedwith pathologies that generate such inflammatory states.

Thus, in certain embodiments, this invention contemplates administeringone or more of the peptides of this invention to a subject at risk for,or incurring, an acute inflammatory response and/or at risk for orincurring a symptom of atherosclerosis.

Thus, for example, a person having or at risk for coronary disease mayprophylactically be administered a polypeptide of this invention duringflu season. A person (or animal) subject to a recurrent inflammatorycondition, e.g. rheumatoid arthritis, various autoimmune diseases, etc.,can be treated with a polypeptide of this invention to mitigate orprevent the development of atherosclerosis or stroke. A person (oranimal) subject to trauma, e.g. acute injury, tissue transplant, etc.can be treated with a polypeptide of this invention to mitigate thedevelopment of atherosclerosis or stroke.

In certain instances such methods will entail a diagnosis of theoccurrence or risk of an acute inflammatory response. The acuteinflammatory response typically involves alterations in metabolism andgene regulation in the liver. It is a dynamic homeostatic process thatinvolves all of the major systems of the body, in addition to theimmune, cardiovascular and central nervous system. Normally, the acutephase response lasts only a few days; however, in cases of chronic orrecurring inflammation, an aberrant continuation of some aspects of theacute phase response may contribute to the underlying tissue damage thataccompanies the disease, and may also lead to further complications, forexample cardiovascular diseases or protein deposition diseases such asamyloidosis.

An important aspect of the acute phase response is the radically alteredbiosynthetic profile of the liver. Under normal circumstances, the liversynthesizes a characteristic range of plasma proteins at steady stateconcentrations. Many of these proteins have important functions andhigher plasma levels of these acute phase reactants (APRs) or acutephase proteins (APPs) are required during the acute phase responsefollowing an inflammatory stimulus. Although most APRs are synthesizedby hepatocytes, some are produced by other cell types, includingmonocytes, endothelial cells, fibroblasts and adipocytes. Most APRs areinduced between 50% and several-fold over normal levels. In contrast,the major APRs can increase to 1000-fold over normal levels. This groupincludes serum amyloid A (SAA) and either C-reactive protein (CRP) inhumans or its homologue in mice, serum amyloid P component (SAP).So-called negative APRs are decreased in plasma concentration during theacute phase response to allow an increase in the capacity of the liverto synthesize the induced APRs.

In certain embodiments, the acute phase response, or risk therefore isevaluated by measuring one or more APPs. Measuring such markers is wellknown to those of skill in the art, and commercial companies exist thatprovide such measurement (e.g. AGP measured by Cardiotech Services,Louisville, Ky.).

VII. Mitigation of a Symptom or Condition Associated with CoronaryCalcification and Osteoporosis.

We have also identified oxidized lipids as a cause of coronarycalcification and osteoporosis. Moreover, without being bound to aparticularly theory, we believe the same mechanisms are involved in thepathogenesis of calcific aortic stenosis.

Thus, in certain embodiments, this invention contemplates the use of thepeptides described herein to inhibit or prevent a symptom of a diseasesuch as polymyalgia rheumatica, polyarteritis nodosa, scleroderma, lupuserythematosus, idiopathic pulmonary fibrosis, chronic obstructivepulmonary disease, Alzheimers Disease, AIDS, coronary calcification,calcific aortic stenosis, osteoporosis, and the like.

V. Peptide Administration.

The methods of this invention typically involve administering to anorganism, preferably a mammal, more preferably a human one or more ofthe peptides of this invention (or mimetics of such peptides). Thepeptide(s) can be administered, as described herein, according to any ofa number of standard methods including, but not limited to injection,suppository, nasal spray, time-release implant, transdermal patch, andthe like. In one particularly preferred embodiment, the peptide(s) areadministered orally (e.g. as a syrup, capsule, or tablet).

The methods can involve the administration of a single peptide of thisinvention or the administration of two or more different peptides. Thepeptides can be provided as monomers or in dimeric, oligomeric orpolymeric forms. In certain embodiments, the multimeric forms maycomprise associated monomers (e.g. ionically or hydrophobically linked)while certain other multimeric forms comprise covalently linked monomers(directly linked or through a linker).

While the invention is described with respect to use in humans, it isalso suitable for animal, e.g. veterinary use. Thus preferred organismsinclude, but are not limited to humans, non-human primates, canines,equines, felines, porcines, ungulates, largomorphs, and the like.

The methods of this invention are not limited to humans or non-humananimals showing one or more symptom(s) of atherosclerosis (e.g.hypertension, plaque formation and rupture, reduction in clinical eventssuch as heart attack, angina, or stroke, high levels of plasmacholesterol, high levels of low density lipoprotein, high levels of verylow density lipoprotein, or inflammatory proteins such as CRP, etc.),but are useful in a prophylactic context. Thus, the peptides of thisinvention (or mimetics thereof) may be administered to organisms toprevent the onset/development of one or more symptoms ofatherosclerosis. Particularly preferred subjects in this context aresubjects showing one or more risk factors for atherosclerosis (e.g.family history, hypertension, obesity, high alcohol consumption,smoking, high blood cholesterol, high blood triglycerides, elevatedblood LDL, VLDL, IDL, or low HDL, diabetes, or a family history ofdiabetes, high blood lipids, heart attack, angina or stroke, etc.).

The peptides of this invention can also be administered to stimulate theformation and cycling of pre-beta high density lipoprotein-likeparticles and/or to promote reverse lipid transport and detoxification.

The peptides are also useful for administration with statins where theyenhance (e.g., synergize) the activity of the statin and permit thestatin(s) to be administered at lower dosages and/or theanti-inflammatory properties of statins at any given dose aresignificantly enhanced.

In addition, the peptides can be administered to reduce or eliminate oneor more symptoms of osteoporosis and/or to prevent/inhibit the onset ofone or more symptoms of osteoporosis.

VIII. Preferred Peptides and their Preparation.

Preferred Peptides.

It was a discovery of this invention that peptides comprising a class Aamphipathic helix (“class A peptides”), are capable of mitigating one ormore symptoms of atherosclerosis. Class A peptides are characterized byformation of an α-helix that produces a segregation of polar andnon-polar residues thereby forming a polar and a nonpolar face with thepositively charged residues residing at the polar-nonpolar interface andthe negatively charged residues residing at the center of the polar face(see, e.g., Anantharamaiah (1986) Meth. Enzymol, 128: 626-668). It isnoted that the fourth exon of apo A-I, when folded into 3.667residues/turn produces a class A amphipathic helical structure.

One particularly preferred class A peptide, designated 18A (see, 1, andalso Anantharamaiah (1986) Meth. Enzymol, 128: 626-668) was modified asdescribed herein to produce peptides orally administratable and highlyeffective at inhibiting or preventing one or more symptoms ofatherosclerosis. Without being bound by a particular theory, it isbelieved that the peptides of this invention act in vivo may by pickingup seeding molecule(s) that mitigate oxidation of LDL.

We determined that increasing the number of Phe residues on thehydrophobic face of 18A would theoretically increase lipid affinity asdetermined by the computation described by Palgunachari et al. (1996)Arteriosclerosis, Thrombosis, & Vascular Biology 16: 328-338.Theoretically, a systematic substitution of residues in the nonpolarface of 18A with Phe could yield six peptides. Peptides with anadditional 2, 3 and 4 Phe would have theoretical lipid affinity (λ)values of 13, 14 and 15 units, respectively. However, the λ valuesjumped four units if the additional Phe were increased from 4 to 5 (to19 λunits). Increasing to 6 or 7 Phe would produce a less dramaticincrease (to 20 and 21 λunits, respectively). Therefore, we chose 5additional Phe (and hence the peptides designation as 5F). In oneparticularly preferred embodiment, the 5F peptide was blocked in thatthe amino terminal residue was acetylated and the carboxyl terminalresidue was amidated.

The new class A peptide analog, 5F, inhibited lesion development inatherosclerosis-susceptible mice. The new peptide analog, 5F, wascompared with mouse apo A-I (MoA-I) for efficacy in inhibitingdiet-induced atherosclerosis in these mice using peptide dosages basedon the study by Levine et al. (Levine et al. (1993) Proc. Natl. Acad.Sci. USA 90:12040-12044).

A number of other class A peptides were also produced and showedvarying, but significant degrees of efficacy in mitigating one or moresymptoms of atherosclerosis. A number of such peptides are illustratedin Table 1.

TABLE 1 Preferred peptides for use in this invention. Pep- SEQ tide IDName Amino Acid Sequence NO. 18A    D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F1 2F Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH₂ 2 3FAc-D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH₂ 3 3F14Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ 4 4FAc-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ 5 5FAc-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ 6 6FAc-D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH₂ 7 7FAc-D-W-F-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH₂ 8Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH₂ 9Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH₂ 10Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH₂ 11Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH₂ 12Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ 13Ac-E-W-L-K-L-F-Y-E-K-V-L-E-K-F-K-E-A-F-NH₂ 14Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ 15Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH₂ 16Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH₂ 17Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH₂ 18Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH₂ 19Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ 20        AC-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH₂ 21        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ 22        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ 23        Ac-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH₂ 24        Ac-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH₂ 25        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ 26        Ac-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH₂ 27        Ac-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH₂ 28        Ac-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH₂ 29        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH₂ 30        Ac-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-NH₂ 31        Ac-L-F-Y-E-K-V-L-E-K-F-K-E-A-F-NH₂ 32        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ 33        Ac-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH₂ 34        Ac-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH₂ 35        Ac-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH₂ 36        Ac-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH₂ 37        Ac-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ 38Ac-D-W-L-K-A-L-Y-D-K-V-A-E-K-L-K-E-A-L-NH₂ 39Ac-D-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-NH₂ 40Ac-D-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH₂ 41Ac-E-W-L-K-A-L-Y-E-K-V-A-E-K-L-K-E-A-L-NH₂ 42Ac-E-W-L-K-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-NH₂ 43Ac-E-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-NH₂ 44Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH₂ 45Ac-E-W-L-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH₂ 46Ac-E-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH₂ 47Ac-D-F-L-K-A-W-Y-D-K-V-A-E-K-L-K-E-A-W-NH₂ 48Ac-E-F-L-K-A-W-Y-E-K-V-A-E-K-L-K-E-A-W-NH₂ 49Ac-D-F-W-K-A-W-Y-D-K-V-A-E-K-L-K-E-W-W-NH₂ 50Ac-E-F-W-K-A-W-Y-E-K-V-A-E-K-L-K-E-W-W-NH₂ 51Ac-D-K-L-K-A-F-Y-D-K-V-F-E-W-A-K-E-A-F-NH₂ 52Ac-D-K-W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L-NH₂ 53Ac-E-K-L-K-A-F-Y-E-K-V-F-E-W-A-K-E-A-F-NH₂ 54Ac-E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L-NH₂ 55Ac-D-W-L-K-A-F-V-D-K-F-A-E-K-F-K-E-A-Y-NH₂ 56Ac-E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L-NH₂ 57Ac-D-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F-NH₂ 58Ac-E-W-L-K-A-F-V-Y-E-K-V-F-K-L-K-E-F-F-NH₂ 59Ac-D-W-L-R-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH₂ 60Ac-E-W-L-R-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-NH₂ 61Ac-D-W-L-K-A-F-Y-D-R-V-A-E-K-L-K-E-A-F-NH₂ 62Ac-E-W-L-K-A-F-Y-E-R-V-A-E-K-L-K-E-A-F-NH₂ 63Ac-D-W-L-K-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-NH₂ 64Ac-E-W-L-K-A-F-Y-E-K-V-A-E-R-L-K-E-A-F-NH₂ 65Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-R-E-A-F-NH₂ 66Ac-E-W-L-K-A-F-Y-E-K-V-A-E-K-L-R-E-A-F-NH₂ 67Ac-D-W-L-K-A-F-Y-D-R-V-A-E-R-L-K-E-A-F-NH₂ 68Ac-E-W-L-K-A-F-Y-E-R-V-A-E-R-L-K-E-A-F-NH₂ 69Ac-D-W-L-R-A-F-Y-D-K-V-A-E-K-L-R-E-A-F-NH₂ 70Ac-E-W-L-R-A-F-Y-E-K-V-A-E-K-L-R-E-A-F-NH₂ 71Ac-D-W-L-R-A-F-Y-D-R-V-A-E-K-L-K-E-A-F-NH₂ 72Ac-E-W-L-R-A-F-Y-E-R-V-A-E-K-L-K-E-A-F-NH₂ 73Ac-D-W-L-K-A-F-Y-D-K-V-A-E-R-L-R-E-A-F-NH₂ 74Ac-E-W-L-K-A-F-Y-E-K-V-A-E-R-L-R-E-A-F-NH₂ 75Ac-D-W-L-R-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-NH₂ 76Ac-E-W-L-R-A-F-Y-E-K-V-A-E-R-L-K-E-A-F-NH₂ 77D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F -P- D-W- 78L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F -P-D-W- 79 L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-FD-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F -P- D-W- 80F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F D-K-L-K-A-F-Y-D-K-V-F-E-W-A-K-E-A-F -P-D-K- 81 L-K-A-F-Y-D-K-V-F-E-W-L-K-E-A-FD-K-W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L -P- D-K- 82W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F -P-D-W- 83 F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-FD-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F -P- D-W- 84L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F D-W-L-K-A-F-Y-D-K-F-A-E-K-F-K-E-F-F -P-D-W- 85 L-K-A-F-Y-D-K-F-A-E-K-F-K-E-F-FAc-E-W-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-A-F-NH₂ 86Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-NH₂ 87 Ac-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-NH₂88 Ac-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-NH₂ 89NMA-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-NH₂ 90NMA-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-NH₂ 91NMA-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ 92NMA-E-W-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-A-F- 93 NH₂NMA-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ 94NMA-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-NH₂ 95 Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ 96NMA-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ 96 Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH₂ 97NMA-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH₂ 97 Ac-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ 98NMA-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH₂ 98 Ac-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH₂ 99NMA-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH₂ 99 Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-NH₂ 100NMA-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-NH₂ 100Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-NH₂ 101NMA-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-NH₂ 101Ac-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-NH₂ 102NMA-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-NH₂ 102Ac-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-NH₂ 103NMA-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-NH₂ 103 ¹Linkers are underlined. NMA isN-Methyl Anthranilyl.

In certain preferred embodiments, the peptides include variations of 4For D-4F where one or both aspartic acids (D) are replaced by glutamicacid (E). Also contemplated are peptides (e.g. 4F or D-4F) where 1, 2,3, or 4 amino acids are deleted from the carboxyl terminus and/or 1, 2,3, or 4 amino acids are deleted from the carboxyl terminus and/or one orboth aspartic acids (D) are replaced by glutamic acid (E). In any of thepeptides described herein, the N-terminus can be blocked and labeledusing a mantyl moiety (e.g. N-methylanthranilyl).

While various peptides of 1, are illustrated with an acetyl group or anN-methylanthranilyl group protecting the amino terminus and an amidegroup protecting the carboxyl terminus, any of these protecting groupsmay be eliminated and/or substituted with another protecting group asdescribed herein. In particularly preferred embodiments, the peptidescomprise one or more D-form amino acids as described herein. In certainembodiments, every amino acid (e.g. every enantiomeric amino acid) ofthe peptides of Table 1 is a D-form amino acid.

It is also noted that 1 is not fully inclusive. Using the teachingprovided herein, other suitable peptides can routinely be produced (e.g.by conservative or semi-conservative substitutions (e.g. D replaced byE), extensions, deletions, and the like). Thus, for example, oneembodiment utilizes truncations of any one or more of peptidesidentified by SEQ ID Nos: 2-20 and 39-85. Thus, for example, SEQ ID NO:21 illustrates a peptide comprising 14 amino acids from the C-terminusof 18A comprising one or more D amino acids, while SEQ ID NOS: 22-38illustrate other truncations. Longer peptides are also suitable. Suchlonger peptides may entirely form a class A amphipathic helix, or theclass A amphipathic helix (helices) may form one or more domains of thepeptide. In addition, this invention contemplates multimeric versions ofthe peptides. Thus, for example, the peptides illustrated in 1 can becoupled together (directly or through a linker (e.g. a carbon linker, orone or more amino acids) with one or more intervening amino acids).Illustrative polymeric peptides include 18A-Pro-18A and the peptides ofSEQ ID NOs: 79-85 preferably comprising one or more D amino acids, morepreferably with every amino acid a D amino acid as described hereinand/or having one or both termini protected.

It was a surprising discovery of this invention that, when the class Apeptides (e.g. as illustrated in 1) incorporated D amino acids theyretained their activity and, but could be administered orally. Moreoverthis oral administration resulted in relatively efficient uptake andsignificant serum half-life thereby providing an efficacious method ofmitigating one or more symptoms of atherosclerosis.

Using the teaching provided herein, one of skill can routinely modifythe illustrated class A peptides to produce other suitable class Apeptides of this invention. For example, routine conservative orsemi-conservative substitutions (e.g. E for D) can be made of theexisting amino acids. The effect of various substitutions on lipidaffinity of the resulting peptide can be predicted using thecomputational method described by Palgunachari et al. (1996)Arteriosclerosis, Thrombosis, & Vascular Biology 16: 328-338. Thepeptides can be lengthened or shortened as long as the class A α-helixstructure is preserved. In addition, substitutions can be made to renderthe resulting peptide more similar to peptide(s) endogenously producedby the subject species.

In certain embodiments, the peptides of this invention comprise “D”forms of the peptides described in U.S. Pat. No. 4,643,988, morepreferably “D” forms having one or both termini coupled to protectinggroups. Such peptides include peptides having the formulaA₁-B₁-B₂-C₁-D-B₃-B₄-A₂-C₂-B₅-B₆-A₃-C₃-B₇-C₄-A₄-B₈-B₉ (SEQ ID NO: 104)wherein A₁, A₂, A₃ and A₄ are independently aspartic acid or glutamicacid, or homologues or analogues thereof; B₁, B₂, B₃, B₄, B₅, B₆, B₇, B₈and B₉ are independently tryptophan, phenylalanine, alanine, leucine,tyrosine, isoleucine, valine or α-naphthylalanine, or homologues oranalogues thereof; C₁, C₂, C₃ and C₄ are independently lysine orarginine, and D is serine, threonine, alanine, glycine, histidine, orhomologues or analogues thereof; provided that, when A₁ and A₂ areaspartic acid, A₃ and A₄ are glutamic acid, B₂ and B₉ are leucine, B₃and B₇ are phenylalanine, B₄ is tyrosine, B₅ is valine, B₆, B₈, and Dare alanine, and C₁, C₂, C₃ and C₄ are lysine, B₁ is not Tryptophan,where at one enantiomeric amino acid is a “D” form amino acids.Preferably at least 50% of the enantiomeric amino acids are “D” form,more preferably at least 80% of the enantiomeric amino acids are “D”form, and most preferably at least 90% or even all of the enantiomericamino acids are “D” form amino acids.

While, in preferred embodiments, the peptides of this invention utilizenaturally-occurring amino acids or D forms of naturally occurring aminoacids, substitutions with non-naturally occurring amino acids (e.g.,methionine sulfoxide, methionine methylsulfonium, norleucine,episilon-aminocaproic acid, 4-aminobutanoic acid,tetrahydroisoquinoline-3-carboxylic acid, 8-aminocaprylic acid,4-aminobutyric acid, Lys(N(epsilon)-trifluoroacetyl), α-aminoisobutyricacid, and the like) are also contemplated.

In addition to the class A peptides described herein, peptidomimeticsare also contemplated herein. Peptide analogs are commonly used in thepharmaceutical industry as non-peptide drugs with properties analogousto those of the template peptide. These types of non-peptide compoundare termed “peptide mimetics” or “peptidomimetics” (Fauchere (1986) Adv.Drug Res. 15: 29; Veber and Freidinger (1985) TINS p. 392; and Evans etal. (1987) J. Med. Chem. 30: 1229) and are usually developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect.

Generally, peptidomimetics are structurally similar to a paradigmpolypeptide (i.e., 5F described herein), but have one or more peptidelinkages optionally replaced by a linkage selected from the groupconsisting of: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans),—COCH₂—, —CH(OH)CH₂—, —CH₂SO—, etc. by methods known in the art andfurther described in the following references: Spatola (1983) p. 267 inChemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B.Weinstein, eds., Marcel Dekker, New York; Spatola (1983) Vega Data 1(3)Peptide Backbone Modifications. (general review); Morley (1980) TrendsPharm Sci pp. 463-468 (general review); Hudson et al. (1979) Int J PeptProt Res 14:177-185 (—CH₂NH—, CH₂CH₂—); Spatola et al. (1986) Life Sci38:1243-1249 (—CH₂—S); Hann, (1982) J Chem Soc Perkin Trans I 307-314(—CH—CH—, cis and trans); Almquist et al. (1980) J Med Chem.23:1392-1398 (—COCH₂—); Jennings-White et al. (1982) Tetrahedron Lett.23:2533 (—COCH₂—); Szelke, M. et al., European Appln. EP 45665 (1982)CA: 97:39405 (1982) (—CH(OH)CH2-); Holladay et al. (1983) TetrahedronLett 24:4401-4404 (—C(OH)CH₂—); and Hruby (1982) Life Sci., 31:189-199(—CH₂—S—)).

A particularly preferred non-peptide linkage is —CH₂NH—. Such peptidemimetics may have significant advantages over polypeptide embodiments,including, for example: more economical production, greater chemicalstability, enhanced pharmacological properties (half-life, absorption,potency, efficacy, etc.), reduced antigenicity, and others.

In addition, circularly permutations of the peptides described herein orconstrained peptides (including cyclized peptides) comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch (1992) Ann. Rev. Biochem. 61: 387); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

Peptide Preparation.

The peptides used in this invention can be chemically synthesized usingstandard chemical peptide synthesis techniques or, particularly wherethe peptide does not comprise “D” amino acid residues, the peptide canreadily be recombinantly expressed. Where the “D” polypeptides arerecombinantly expressed, a host organism (e.g. bacteria, plant, fungalcells, etc.) can be cultured in an environment where one or more of theamino acids is provided to the organism exclusively in a D form.Recombinantly expressed peptides in such a system then incorporate thoseD amino acids.

In certain embodiments, D amino acids can be incorporated inrecombinantly expressed peptides using modified amino acyl-tRNAsynthetases that recognize D-amino acids.

In certain preferred embodiments the peptides are chemically synthesizedby any of a number of fluid or solid phase peptide synthesis techniquesknown to those of skill in the art. Solid phase synthesis in which theC-terminal amino acid of the sequence is attached to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence is a preferred method for the chemical synthesis of thepolypeptides of this invention. Techniques for solid phase synthesis arewell known to those of skill in the art and are described, for example,by Barany and Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptideSynthesis, Part A.; Merrifield et al. (1963) J. Am. Chem. Soc.,85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis,2nd ed. Pierce Chem. Co., Rockford, Ill.

In one embodiment, the peptides are synthesized by the solid phasepeptide synthesis procedure using a benzhyderylamine resin (BeckmanBioproducts, 0.59 mmol of NH₂/g of resin) as the solid support. The COOHterminal amino acid (e.g., t-butylcarbonyl-Phe) is attached to the solidsupport through a 4-(oxymethyl)phenacetyl group. This is a more stablelinkage than the conventional benzyl ester linkage, yet the finishedpeptide can still be cleaved by hydrogenation. Transfer hydrogenationusing formic acid as the hydrogen donor is used for this purpose.Detailed protocols used for peptide synthesis and analysis ofsynthesized peptides are describe in a miniprint supplement accompanyingAnantharamaiah et al. (1985) J. Biol. Chem., 260(16): 10248-10255.

It is noted that in the chemical synthesis of peptides, particularlypeptides comprising D amino acids, the synthesis usually produces anumber of truncated peptides in addition to the desired full-lengthproduct. The purification process (e.g. HPLC) typically results in theloss of a significant amount of the full-length product.

It was a discovery of this invention that, particularly in the synthesisof a D peptide (e.g. D-4), in order to prevent loss in purifying thelongest form one can dialyze and use the mixture and thereby eliminatethe last HPLC purification. Such a mixture loses about 50% of thepotency of the highly purified product (e.g. per wt of protein product),but the mixture contains about 6 times more peptide and thus greatertotal activity.

D-Form Amino Acids.

D-amino acids can be incorporated at one or more positions in thepeptide simply by using a D-form derivatized amino acid residue in thechemical synthesis. D-form residues for solid phase peptide synthesisare commercially available from a number of suppliers (see, e.g.,Advanced Chem Tech, Louisville; Nova Biochem, San Diego; Sigma, StLouis; Bachem California Inc., Torrance, etc.). The D-form amino acidscan be completely omitted or incorporated at any position in the peptideas desired. Thus, for example, in certain embodiments, the peptide cancomprise a single D-amino acid, while in other embodiments, the peptidecomprises at least two, generally at least three, more generally atleast four, most generally at least five, preferably at least six, morepreferably at least seven and most preferably at least eight D aminoacids. In particularly preferred embodiments, essentially every other(enantiomeric) amino acid is a D-form amino acid. In certain embodimentsat least 90%, preferably at least 90%, more preferably at least 95% ofthe enantiomeric amino acids are D-form amino acids. In one particularlypreferred embodiment, essentially every enantiomeric amino acid is aD-form amino acid.

Protecting Groups.

In certain embodiments, the one or more R-groups on the constituentamino acids and/or the terminal amino acids are blocked with aprotecting group. Without being bound by a particular theory, it was adiscovery of this invention that blockage, particularly of the aminoand/or carboxyl termini of the subject peptides of this inventiongreatly improves oral delivery and significantly increases serumhalf-life.

A wide number of protecting groups are suitable for this purpose. Suchgroups include, but are not limited to acetyl, amide, and alkyl groupswith acetyl and alkyl groups being particularly preferred for N-terminalprotection and amide groups being preferred for carboxyl terminalprotection. In certain embodiments, the blocking groups can additionallyact as a detectable label (e.g. N-methyl anthranilyl).

In certain particularly preferred embodiments, the protecting groupsinclude, but are not limited to alkyl chains as in fatty acids,propionyl, formyl, and others. Particularly preferred carboxylprotecting groups include amides, esters, and ether-forming protectinggroups. In one preferred embodiment, an acetyl group is used to protectthe amino terminus and an amide group is used to protect the carboxylterminus. These blocking groups enhance the helix-forming tendencies ofthe peptides. Certain particularly preferred blocking groups includealkyl groups of various lengths, e.g. groups having the formula:CH₃—(CH₂)_(n)—CO— where n ranges from about 1 to about 20, preferablyfrom about 1 to about 16 or 18, more preferably from about 3 to about13, and most preferably from about 3 to about 10.

In certain particularly preferred embodiments, the protecting groupsinclude, but are not limited to alkyl chains as in fatty acids,propionyl, formyl, and others. Particularly preferred carboxylprotecting groups include amides, esters, and ether-forming protectinggroups. In one preferred embodiment, an acetyl group is used to protectthe amino terminus and an amide group is used to protect the carboxylterminus. These blocking groups enhance the helix-forming tendencies ofthe peptides. Certain particularly preferred blocking groups includealkyl groups of various lengths, e.g. groups having the formula:CH₃—(CH₂)_(n)—CO— where n ranges from about 3 to about 20, preferablyfrom about 3 to about 16, more preferably from about 3 to about 13, andmost preferably from about 3 to about 10.

Other protecting groups include, but are not limited to N-methylanthranilyl, Fmoc, t-butoxycarbonyl (t-BOC), 9-fluoreneacetyl group,1-fluorenecarboxylic group, 9-florenecarboxylic group,9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan),Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt),4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl(Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl(MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), cyclohexyloxy(cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl(Ac), and Trifluoroacetyl (TFA).

Protecting/blocking groups are well known to those of skill as aremethods of coupling such groups to the appropriate residue(s) comprisingthe peptides of this invention (see, e.g., Greene et al., (1991)Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc.Somerset, N.J.). In one preferred embodiment, for example, acetylationis accomplished during the synthesis when the peptide is on the resinusing acetic anhydride. Amide protection can be achieved by theselection of a proper resin for the synthesis. During the synthesis ofthe peptides described herein in the examples, rink amide resin wasused. After the completion of the synthesis, the semipermanentprotecting groups on acidic bifunctional amino acids such as Asp and Gluand basic amino acid Lys, hydroxyl of Tyr are all simultaneouslyremoved. The peptides released from such a resin using acidic treatmentcomes out with the n-terminal protected as acetyl and the carboxylprotected as NH₂ and with the simultaneous removal of all of the otherprotecting groups.

IX. Enhancing Peptide Uptake.

It was also a surprising discovery of this invention that when an all Lamino acid peptide (e.g. otherwise having the sequence of the peptidesof this invention) is administered in conjunction with the D-form (i.e.a peptide of this invention) the uptake of the D-form peptide isincreased. Thus, in certain embodiments, this invention contemplates theuse of combinations of D-form and L-form peptides in the methods of thisinvention. The D-form peptide and the L-form peptide can have differentamino acid sequences, however, in preferred embodiments, they both haveamino acid sequences of peptides described herein, and in still morepreferred embodiments, they have the same amino acid sequence.

It was also a discovery of this invention that concatamers of the classA amphipathic helix peptides of this invention are also effective inmitigating one or more symptoms of atherosclerosis. The monomerscomprising the concatamers can be coupled directly together or joined bya linker. In certain embodiments, the linker is an amino acid linker(e.g. a proline), or a peptide linker (e.g. Gly₄Ser₃, SEQ ID NO: 106).In certain embodiments, the concatamer is a 2 mer, more preferably a 3mer, still more preferably a 4 mer, and most preferably 5 mer, 8 mer or10 mer.

X. Pharmaceutical Formulations.

In order to carry out the methods of the invention, one or more peptidesor peptide mimetics of this invention are administered, e.g. to anindividual diagnosed as having one or more symptoms of atherosclerosis,or as being at risk for atherosclerosis. The peptides or peptidemimetics can be administered in the “native” form or, if desired, in theform of salts, esters, amides, prodrugs, derivatives, and the like,provided the salt, ester, amide, prodrug or derivative is suitablepharmacologically, i.e., effective in the present method. Salts, esters,amides, prodrugs and other derivatives of the active agents may beprepared using standard procedures known to those skilled in the art ofsynthetic organic chemistry and described, for example, by March (1992)Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed.N.Y. Wiley-Interscience.

For example, acid addition salts are prepared from the free base usingconventional methodology, that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or may be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include both organic acids, e.g., aceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malicacid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. An acid addition salt may be reconvertedto the free base by treatment with a suitable base. Particularlypreferred acid addition salts of the active agents herein are halidesalts, such as may be prepared using hydrochloric or hydrobromic acids.Conversely, preparation of basic salts of the peptides or mimetics areprepared in a similar manner using a pharmaceutically acceptable basesuch as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine, or the like. Particularly preferredbasic salts include alkali metal salts, e.g., the sodium salt, andcopper salts.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups which may be present within the molecularstructure of the drug. The esters are typically acyl-substitutedderivatives of free alcohol groups, i.e., moieties that are derived fromcarboxylic acids of the formula RCOOH where R is alky, and preferably islower alkyl. Esters can be reconverted to the free acids, if desired, byusing conventional hydrogenolysis or hydrolysis procedures.

Amides and prodrugs may also be prepared using techniques known to thoseskilled in the art or described in the pertinent literature. Forexample, amides may be prepared from esters, using suitable aminereactants, or they may be prepared from an anhydride or an acid chlorideby reaction with ammonia or a lower alkyl amine. Prodrugs are typicallyprepared by covalent attachment of a moiety that results in a compoundthat is therapeutically inactive until modified by an individual'smetabolic system.

The peptides or mimetics identified herein are useful for parenteral,topical, oral, nasal (or otherwise inhaled), rectal, or localadministration, such as by aerosol or transdermally, for prophylacticand/or therapeutic treatment of atherosclerosis and/or symptoms thereof.The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. Suitable unitdosage forms, include, but are not limited to powders, tablets, pills,capsules, lozenges, suppositories, patches, nasal sprays, injectables,implantable sustained-release formulations, lipid complexes, etc.

The peptides and/or peptide mimetics of this invention are typicallycombined with a pharmaceutically acceptable carrier (excipient) to forma pharmacological composition. Pharmaceutically acceptable carriers cancontain one or more physiologically acceptable compound(s) that act, forexample, to stabilize the composition or to increase or decrease theabsorption of the active agent(s). Physiologically acceptable compoundscan include, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, protection and uptake enhancerssuch as lipids, compositions that reduce the clearance or hydrolysis ofthe active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysio-chemical characteristics of the active agent(s).

The excipients are preferably sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well-knownsterilization techniques.

In therapeutic applications, the compositions of this invention areadministered to a patient suffering from one or more symptoms ofatherosclerosis or at risk for atherosclerosis in an amount sufficientto cure or at least partially prevent or arrest the disease and/or itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. In any event, the composition shouldprovide a sufficient quantity of the active agents of the formulationsof this invention to effectively treat (ameliorate one or more symptoms)the patient.

The concentration of peptide or mimetic can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs. Concentrations, however, willtypically be selected to provide dosages ranging from about 0.1 or 1mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosagesrange from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably fromabout 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about7.2 mg/kg/day to about 11.0 mg/kg/day, and most preferably from about11.0 mg/kg/day to about 15.0 mg/kg/day. In certain preferredembodiments, dosages range from about 10 mg/kg/day to about 50mg/kg/day. It will be appreciated that such dosages may be varied tooptimize a therapeutic regimen in a particular subject or group ofsubjects.

In certain preferred embodiments, the peptides or peptide mimetics ofthis invention are administered orally (e.g. via a tablet) or as aninjectable in accordance with standard methods well known to those ofskill in the art. In other preferred embodiments, the peptides, may alsobe delivered through the skin using conventional transdermal drugdelivery systems, i.e., transdermal “patches” wherein the activeagent(s) are typically contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the drug composition is typically contained in a layer, or“reservoir,” underlying an upper backing layer. It will be appreciatedthat the term “reservoir” in this context refers to a quantity of“active ingredient(s)” that is ultimately available for delivery to thesurface of the skin. Thus, for example, the “reservoir” may include theactive ingredient(s) in an adhesive on a backing layer of the patch, orin any of a variety of different matrix formulations known to those ofskill in the art. The patch may contain a single reservoir, or it maycontain multiple reservoirs.

In one embodiment, the reservoir comprises a polymeric matrix of apharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

Other preferred formulations for topical drug delivery include, but arenot limited to, ointments and creams. Ointments are semisolidpreparations which are typically based on petrolatum or other petroleumderivatives. Creams containing the selected active agent, are typicallyviscous liquid or semisolid emulsions, often either oil-in-water orwater-in-oil. Cream bases are typically water-washable, and contain anoil phase, an emulsifier and an aqueous phase. The oil phase, alsosometimes called the “internal” phase, is generally comprised ofpetrolatum and a fatty alcohol such as cetyl or stearyl alcohol; theaqueous phase usually, although not necessarily, exceeds the oil phasein volume, and generally contains a humectant. The emulsifier in a creamformulation is generally a nonionic, anionic, cationic or amphotericsurfactant. The specific ointment or cream base to be used, as will beappreciated by those skilled in the art, is one that will provide foroptimum drug delivery. As with other carriers or vehicles, an ointmentbase should be inert, stable, nonirritating and nonsensitizing.

Unlike typical peptide formulations, the peptides of this inventioncomprising D-form amino acids can be administered, even orally, withoutprotection against proteolysis by stomach acid, etc. Nevertheless, incertain embodiments, peptide delivery can be enhanced by the use ofprotective excipients. This is typically accomplished either bycomplexing the polypeptide with a composition to render it resistant toacidic and enzymatic hydrolysis or by packaging the polypeptide in anappropriately resistant carrier such as a liposome. Means of protectingpolypeptides for oral delivery are well known in the art (see, e.g.,U.S. Pat. No. 5,391,377 describing lipid compositions for oral deliveryof therapeutic agents).

Sustained Release Formulations.

Elevated serum half-life can be maintained by the use ofsustained-release protein “packaging” systems. Such sustained releasesystems are well known to those of skill in the art. In one preferredembodiment, the ProLease biodegradable microsphere delivery system forproteins and peptides (Tracy (1998) Biotechnol. Prog. 14: 108; Johnsonet al. (1996), Nature Med. 2: 795; Herbert et al. (1998), Pharmaceut.Res. 15, 357) a dry powder composed of biodegradable polymericmicrospheres containing the protein in a polymer matrix that can becompounded as a dry formulation with or without other agents.

The ProLease microsphere fabrication process was specifically designedto achieve a high protein encapsulation efficiency while maintainingprotein integrity. The process consists of (i) preparation offreeze-dried protein particles from bulk protein by spray freeze-dryingthe drug solution with stabilizing excipients, (ii) preparation of adrug-polymer suspension followed by sonication or homogenization toreduce the drug particle size, (iii) production of frozen drug-polymermicrospheres by atomization into liquid nitrogen, (iv) extraction of thepolymer solvent with ethanol, and (v) filtration and vacuum drying toproduce the final dry-powder product. The resulting powder contains thesolid form of the protein, which is homogeneously and rigidly dispersedwithin porous polymer particles. The polymer most commonly used in theprocess, poly(lactide-co-glycolide) (PLG), is both biocompatible andbiodegradable.

Encapsulation can be achieved at low temperatures (e.g., −40° C.).During encapsulation, the protein is maintained in the solid state inthe absence of water, thus minimizing water-induced conformationalmobility of the protein, preventing protein degradation reactions thatinclude water as a reactant, and avoiding organic-aqueous interfaceswhere proteins may undergo denaturation. A preferred process usessolvents in which most proteins are insoluble, thus yielding highencapsulation efficiencies (e.g., greater than 95%).

In another embodiment, one or more components of the solution can beprovided as a “concentrate”, e.g., in a storage container (e.g., in apremeasured volume) ready for dilution, or in a soluble capsule readyfor addition to a volume of water.

Combined Formulations.

In certain instances, one or more peptides of this invention areadministered in conjunction with one or more active agents (e.g.statins, beta blockers, ACE inhibitors, lipids, etc.). The two agents(e.g. peptide and statin) can be administered simultaneously orsequentially. When administered sequentially the two agents areadministered so that both achieve a physiologically relevantconcentration over a similar time period (e.g. so that both agents areactive at some common time).

In certain embodiments, both agents are administered simultaneously. Insuch instances it can be convenient to provide both agents in a singlecombined formulation. This can be achieved by a variety of methods wellknown to those of skill in the art. For example, in a table formulationthe table can comprise two layers one layer comprising, e.g. thestatin(s), and the other layer comprising e.g. the peptide(s). In a timerelease capsule, the capsule can comprise two time release bead sets,one for the peptide(s) and one containing the statin(s).

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

XI. Additional Pharmacologically Active Agents.

Additional pharmacologically active agents may be delivered along withthe primary active agents, e.g., the peptides of this invention. In oneembodiment, such agents include, but are not limited to agents thatreduce the risk of atherosclerotic events and/or complications thereof.Such agents include, but are not limited to beta blockers, beta blockersand thiazide diuretic combinations, statins, aspirin, ace inhibitors,ace receptor inhibitors (ARBs), and the like.

Statins.

It was a surprising discovery that administration of one or morepeptides of this invention “concurrently” with one or more statinssynergistically enhances the effect of the statin(s). That is, thestatins can achieve a similar efficacy at lower dosage thereby obviatingpotential adverse side effects (e.g. muscle wasting) associated withthese drugs and/or cause the statins to be significantly moreanti-inflammatory at any given dose.

The major effect of the statins is to lower LDL-cholesterol levels, andthey lower LDL-cholesterol more than many other types of drugs. Statinsgenerally inhibit an enzyme, HMG-CoA reductase, that controls the rateof cholesterol production in the body. These drugs typically lowercholesterol by slowing down the production of cholesterol and byincreasing the liver's ability to remove the LDL-cholesterol already inthe blood.

The large reductions in total and LDL-cholesterol produced by thesedrugs appears to result in large reductions in heart attacks and heartdisease deaths. Thanks to their track record in these studies and theirability to lower LDL-cholesterol, statins have become the drugs mostoften prescribed when a person needs a cholesterol-lowering medicine.Studies using statins have reported 20 to 60 percent lowerLDL-cholesterol levels in patients on these drugs. Statins also reduceelevated triglyceride levels and produce a modest increase inHDL-cholesterol. Recently it has been appreciated that statins haveanti-inflammatory properties that may not be directly related to thedegree of lipid lowering achieved. For example it has been found thatstatins decrease the plasma levels of the inflammatory marker CRPrelatively independent of changes in plasma lipid levels. Thisanti-inflammatory activity of statins has been found to be as or moreimportant in predicting the reduction in clinical events induced bystatins than is the degree of LDL lowering.

The statins are usually given in a single dose at the evening meal or atbedtime. These medications are often given in the evening to takeadvantage of the fact that the body makes more cholesterol at night thanduring the day. When combined with the peptides described herein, thecombined peptide/statin treatment regimen will also typically be givenin the evening.

Suitable statins are well known to those of skill in the art. Suchstatins include, but are not limited to atorvastatin (Lipitor®, Pfizer),simvastatin (Zocor®, Merck0, pravastatin (Pravachol®, Bristol-MyersSquibb0, fluvastatin (Lescol®, Novartis), lovastatin (Mevacor®, Merck),rosuvastatin (Crestor®, Astra Zeneca), and Pitavastatin (Sankyo), andthe like.

The combined statin/peptide dosage can be routinely optimized for eachpatient. Typically statins show results after several weeks, with amaximum effect in 4 to 6 weeks. Prior to combined treatment with astatin and one of the peptides described herein, the physician wouldobtain routine tests for starting a statin including LDL-cholesterol andHDL-cholesterol levels. Additionally, the physician would also measurethe anti-inflammatory properties of the patient's HDL and determine CRPlevels with a high sensitivity assay. After about 4 to 6 weeks ofcombined treatment, the physician would typically repeat these tests andadjust the dosage of the medications to achieve maximum lipid loweringand maximum anti-inflammatory activity.

Beta Blockers.

Suitable beta blockers include, but are not limited to cardioselective(selective beta 1 blockers), e.g., acebutolol (Sectral™), atenolol(Tenormin™), betaxolol (Kerlone™), bisoprolol (Zebeta™), metoprolol(Lopressor™), and the like. Suitable non-selective blockers (block beta1 and beta 2 equally) include, but are not limited to carteolol(Cartrol™), nadolol (Corgard™), penbutolol (Levatol™), pindolol(Visken™), propranolol (Inderal™), timolol (Blockadren™), labetalol(Normodyne™, Trandate™), and the like.

Suitable beta blocker thiazide diuretic combinations include, but arenot limited to Lopressor HCT, ZIAC, Tenoretic, Corzide, Timolide,Inderal LA 40/25, Inderide, Normozide, and the like.

ACE Inhibitors.

Suitable ace inhibitors include, but are not limited to captopril (e.g.Capoten™ by Squibb), benazepril (e.g., Lotensin™ by Novartis), enalapril(e.g., Vasotec™ by Merck), fosinopril (e.g., Monopril™ byBristol-Myers), lisinopril (e.g. Prinivil™ by Merck or Zestril™ byAstra-Zeneca), quinapril (e.g. Accupril™ by Parke-Davis), ramipril(e.g., Altace™ by Hoechst Marion Roussel, King Pharmaceuticals),imidapril, perindopril erbumine (e.g., Aceon™ by Rhone-Polenc Rorer),trandolapril (e.g., Mavik™ by Knoll Pharmaceutical), and the like.Suitable ARBS (Ace Receptor Blockers) include but are not limited tolosartan (e.g. Cozaar™ by Merck), irbesartan (e.g., Avapro™ by Sanofi),candesartan (e.g., Atacand™ by Astra Merck), valsartan (e.g., Diovan™ byNovartis), and the like.

Lipid-Based Formulations.

In certain embodiments, the peptides of this invention are administeredin conjunction with one or more lipids. The lipids can be formulated asan active agent, and/or as an excipient to protect and/or enhancetransport/uptake of the peptides or they can be administered separately.

Without being bound by a particular theory, it was discovered of thisinvention that administration (e.g. oral administration) of certainphospholipids can significantly increase HDL/LDL ratios. In addition, itis believed that certain medium-length phospholipids are transported bya process different than that involved in general lipid transport. Thus,co-administration of certain medium-length phospholipids with thepeptides of this invention confer a number of advantages: They protectthe phospholipids from digestion or hydrolysis, they improve peptideuptake, and they improve HDL/LDL ratios.

The lipids can be formed into liposomes that encapsulate thepolypeptides of this invention and/or they can be simplycomplexed/admixed with the polypeptides. Methods of making liposomes andencapsulating reagents are well known to those of skill in the art (see,e.g., Martin and Papahadjopoulos (1982) J. Biol. Chem., 257: 286-288;Papahadjopoulos et al. (1991) Proc. Natl. Acad. Sci. USA, 88:11460-11464; Huang et al. (1992) Cancer Res., 52:6774-6781; Lasic et al.(1992) FEBS Lett., 312: 255-258., and the like).

Preferred phospholipids for use in these methods have fatty acidsranging from about 4 carbons to about 24 carbons in the sn-1 and sn-2positions. In certain preferred embodiments, the fatty acids aresaturated. In other preferred embodiments, the fatty acids can beunsaturated. Various preferred fatty acids are illustrated in Table 2.

TABLE 2 Preferred fatty acids in the sn-1 and/or sn-2 position of thepreferred phospholipids for administration of D polypeptides. Carbon No.Common Name IUPAC Name  3:0 Propionoyl Trianoic  4:0 Butanoyl Tetranoic 5:0 Pentanoyl Pentanoic  6:0 Caproyl Hexanoic  7:0 Heptanoyl Heptanoic 8:0 Capryloyl Octanoic  9:0 Nonanoyl Nonanoic 10:0 Capryl Decanoic 11:0Undcanoyl Undecanoic 12:0 Lauroyl Dodecanoic 13:0 TridecanoylTridecanoic 14:0 Myristoyl Tetradecanoic 15:0 PentadecanoylPentadecanoic 16:0 Palmitoyl Hexadecanoic 17:0 HeptadecanoylHeptadecanoic 18:0 Stearoyl Octadecanoic 19:0 Nonadecanoyl Nonadecanoic20:0 Arachidoyl Eicosanoic 21:0 Heniecosanoyl Heniecosanoic 22:0Behenoyl Docosanoic 23:0 Trucisanoyl Trocosanoic 24:0 LignoceroylTetracosanoic 14:1 Myristoleoyl (9-cis) 14:1 Myristelaidoyl (9-trans)16:1 Palmitoleoyl (9-cis) 16:1 Palmitelaidoyl (9-trans)

The fatty acids in these positions can be the same or different.Particularly preferred phospholipids have phosphorylcholine at the sn-3position.

XII. Kits.

In another embodiment this invention provides kits for amelioration ofone or more symptoms of atherosclerosis and/or for the prophylactictreatment of a subject (human or animal) at risk for atherosclerosisand/or for stimulating the formation and cycling of pre-beta highdensity lipoprotein-like particles and/or for inhibiting one or moresymptoms of osteoporosis. The kits preferably comprise a containercontaining one or more of the peptides or peptide mimetics of thisinvention. The peptide or peptide mimetic can be provided in a unitdosage formulation (e.g. suppository, tablet, caplet, patch, etc.)and/or may be optionally combined with one or more pharmaceuticallyacceptable excipients.

The kit can, optionally, further comprise one or more other agents usedin the treatment of heart disease and/or atherosclerosis. Such agentsinclude, but are not limited to, beta blockers, vasodilators, aspirin,statins, ace inhibitors or ace receptor inhibitors (ARBs) and the like,e.g. as described above.

In certain preferred embodiments, the kits additionally include a statin(e.g. cerivastatin, atorvastatin, simvastatin, pravastatin, fluvastatin,lovastatin, rosuvastatin, pitavastatin, etc.) either formulatedseparately or in a combined formulation with the peptide(s). Typicallythe dosage of a statin in such a formulation can be lower than thedosage of a statin typically prescribed without the synergistic peptide.

In addition, the kits optionally include labeling and/or instructionalmaterials providing directions (i.e., protocols) for the practice of themethods or use of the “therapeutics” or “prophylactics” of thisinvention. Preferred instructional materials describe the use of one ormore polypeptides of this invention to mitigate one or more symptoms ofatherosclerosis and/or to prevent the onset or increase of one or moreof such symptoms in an individual at risk for atherosclerosis and/or tostimulate the formation and cycling of pre-beta high densitylipoprotein-like particles and/or to inhibit one or more symptoms ofosteoporosis. The instructional materials may also, optionally, teachpreferred dosages/therapeutic regiment, counter indications and thelike.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Lipid Transport and Detoxification by Stimulating theFormation and Cycling of Pre-Beta High Density Lipoprotein-LikeParticles

The serum concentration of D-4F after oral administration was comparedto that obtained by injection. FIG. 1 shows the results ofadministration of ¹⁴C-D-4F to apoE null mice by stomach tube. Bloodsamples were obtained at each time point shown in FIG. 1.

FIG. 2 demonstrates the radioactivity in plasma after the same amount ofmass and radioactivity of D-4F was given to the mice by tail veininjection. The data in FIGS. 1 and 2 indicate that 2 hours after oraladministration, about 1.2% of the radioactivity seen after IV injectionwas present in plasma. From a number of studies we have concluded thatapproximately 1% of an oral dose of D-4F is absorbed.

Based on the low absorption, one might think that D-4F would not bebiologically active. Normal mouse plasma contains on the order of 100mg/dl or 1 mg/ml of apoAI. Since apoE null mice have low HDL-cholesterollevels, if we assume that their apoA-I levels are only one-fifth normal,they would have about. 0.20 mg/ml of apoA-I. If we instilled 500 μg ofD-4F into the stomach of a mouse and only 1% was absorbed, we would haveadded 5 μg of D-4F to approximately 1.5 ml of mouse plasma so we wouldhave approximately 3.3 μg of D-4F per ml of mouse plasma. So how could3.3 μg of D-4F per ml of mouse plasma influence lipid transport whenapoE null mouse plasma already contains approximately 200 μg of apoA-I.

In other studies, 10 minutes after instilling either saline or 500 μg[¹⁴C]-D-4F into the stomachs of apoE null mice there was no detectableD-4F in plasma. However, 20 minutes after D-4F was given, it wasdetected in plasma. When the plasma was fractionated by FPLC the HDLpeak eluted in fraction 30 and the D-4F eluted in FPLC fractions 35 to37 (labeled CCP in FIG. 3). In the absence of D-4F (no D-4F), fraction35 contained some apoA-I by Western blotting, fraction 36 contained lessapoA-I and fraction 37 contained almost no detectable apoA-I (FIG. 3).Moreover, in the absence of D-4F the size of the particles containingapoA-I was approximately 10.5 nm by PAGE (FIG. 3). Twenty min aftergiving D-4F (+D-4F), by Western blotting, fraction 35 contained someapoA-I, fraction 36 contained more apoA-I, and fraction 37 containedmore apoA-I than in fraction 35, but less than in fraction 36 (FIG. 3).However after D-4F, the particles containing apoA-I in fractions 35-37were smaller (8.5 vs 10.5 nm) (see +D-4F, CCP in FIG. 3).

By LC-MRM, 20 min after giving D-4F orally, fractions 35-37 were alsoseen to contain D-4F (FIG. 4). These results were also confirmed with¹⁴C-D-4F. Additionally, after giving D-4F orally, the small particles infractions 35-37 containing apoA-I also contained paraoxonase activityand cholesterol (FIG. 5, bottom panel). In the absence of D-4F,fractions 35-37 contained virtually no paraoxonase activity and verylittle if any cholesterol (FIG. 5 top panel).

The bottom panel of FIG. 5 shows the fractions with cholesterolcontaining particles (CCP) that appeared to the right of HDL after D-4F.As shown in the bottom panel of FIG. 5, the CCP fractions also containedparaoxonase (PON) activity after D-4F.

As indicated in FIG. 3, after giving D-4F orally and analyzing fractions35-37 by PAGE these particles were approximately 8.5 nm in size. Whenthese fractions were purified with preparative PAGE and examined bynegative staining electron microscopy, their size was determined to be 8to 9 nm confirming the estimation by PAGE. By 6.5 hours after givingD-4F orally, almost all of the D-4F had moved to larger lipoproteinparticles with an affinity for HDL that was more than 10-fold greaterthan for B containing lipoproteins. Twenty min after ¹⁴C-D-4F was givenby tail vein injection, there was more than 100-fold more D-4F in plasmaas compared to after oral administration by stomach tube. However, thecontent of D-4F in FPLC fractions 35-37 was only about 2-fold greaterafter injection as compared to after oral administration (data notshown).

We conclude from these data that 20 min after absorption from theintestine D-4F forms small pre-beta HDL-like particles that containrelatively high amounts of apoA-I and paraoxonase. Indeed, estimatingthe amount of apoA-I in these pre-beta HDL-like particles from Westernblots and comparing the amount of apoA-I to the amount of D-4F in theseparticles (determined by radioactivity or LC-MRM) suggests that as D-4Fis absorbed from the intestine, it acts as a catalyst causing theformation of these pre-beta HDL-like particles. This small amount ofintestinally derived D-4F somehow recruits amounts of apoA-I,paraoxonase, and cholesterol into these particles that are orders ofmagnitude more than the amount of D-4F.

Based on the content of apoA-I, D-4F and paraoxonase in the pre-betaHDL-like particles (CCP) generated 20 min after D-4F (FIGS. 3 to 5), onewould predict that these pre-beta HDL-like particles would detoxifyoxidized lipids and prevent artery wall cells from generating aninflammatory response manifested by the production of monocytechemotactic activity. The experiments shown in FIG. 6 indicate that thiswas indeed the case. As shown in FIG. 6, the pre-beta HDL-like particleregion (CCP) after FPLC separation of plasma from apoE null mice thatdid not receive D-4F promoted the oxidation of PAPC by HPODE and waspro-inflammatory. In contrast, after oral administration of D-4F to theapoE null mice, these fractions prevented the oxidation of PAPC by HPODE(detoxified the lipids) and were anti-inflammatory. Moreover, after D-4Fthe anti-inflammatory properties of the pre-beta HDL-like particles wassimilar to that achieved with a five-fold greater concentration of apoEnull HDL after D-4F. As also shown in FIG. 6 apoE null HDL after D-4Fachieved a degree of lipid detoxification similar to that achieved bynormal human HDL without D-4F at a 7-fold greater concentration. Thus,the pre-beta HDL-like particles in the apoE null mice after D-4F wereapproximately 35-fold better able to detoxify the lipids and prevent theartery wall cells from producing monocyte chemotactic activity than wasnormal human HDL.

To follow the course of these particles over time we labeled D-4F with anovel fluorescent probe as shown in FIG. 7. LDL receptor null femalemice at 8 weeks of age (5 mice per group) were given by stomach tube 22μg/mouse of N-Methyl Anthranilyl-D-4F and were then bleed 1, 2, or 8hours later. Their plasma was fractionated by FPLC and analyzed forcholesterol and fluorescence. The 1-hour time point is shown in FIG. 8A.The 2 hour time point is shown in FIG. 8B, and the 8 hour time point isshown in FIG. 8C.

Following absorption, D-4F rapidly recruits relatively large amounts ofapoA-I and paraoxonase to form pre-beta HDL-like particles which arevery likely the most potent particles for both promoting reversecholesterol transport and for destroying biologically active oxidizedlipids. We believe that the formation of these particles and theirsubsequent rapid incorporation into mature HDL likely explains thedramatic reduction in atherosclerosis that we observed in LDL receptornull mice on a Western diet and in apoE null mice on a chow dietindependent of changes in plasma cholesterol or HDL-cholesterol. Basedon these data, we believe the administration of peptides of thisinvention can promote lipid transport and “detoxification” bystimulating the formation and cycling of pre-beta high densitylipoprotein-like particles.

Example 2 Synergistic Action Between Statins and Orally AdministeredPeptides to Ameliorate Atherosclerosis

ApoE null female mice three months old on a chow diet were givendrinking water alone (Water), or drinking water containing 1 μg/ml ofD-4F, or 0.05 mg/ml of Atorvastatin, or 0.05 mg/ml of Pravastatin, or 1μg/ml of D-4F together with 0.05 mg/ml of Atorvastatin, or 1 μg/ml ofD-4F together with 0.05 mg/ml of Pravastatin. After 24 hours the micewere bled and their HDL was tested in a human artery wall coculturemodel. Twenty μg of1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) wasadded together with 1 μg/ml of hydroperoxyeicosatetraenoic acid (HPODE)to cocultures of human artery wall cells as described previously (Navabet al. (2001) J Lipid Res. 42: 1308-1317). Human HDL (h, HDL) was addedat 350 μg/ml cholesterol or no addition was made to the cocultures (NoAddition), or mouse HDL isolated by FPLC from the mice given drinkingwater alone (Water) or the additions shown on the X-axis were added tothe cocultures at 50 μg/ml HDL-cholesterol. After 8 hours of incubation,supernatants were collected and assayed for monocyte chemotacticactivity using standard neuroprobe chambers.

The results are shown in FIG. 9. As shown in FIG. 9, adding 1 μg/ml ofD-4F to the drinking water of apoE null mice for 24 hours did notsignificantly improve HDL function. FIG. 9 also shows that adding 0.05mg/ml of atorvastatin or pravastatin alone to the drinking water of theapoE null mice for 24 hours did not improve HDL function. However, FIG.9 shows that when D-4F 1 μg/ml was added to the drinking water togetherwith 0.05 mg/ml of atorvastatin or pravastatin there was a significantimprovement in HDL function. Indeed the pro-inflammatory apoE null HDLbecame as anti-inflammatory as 350 μg/ml of normal human HDL (h, HDL)(*=p<0.05).

Thus, doses of D-4F alone, or statins alone, which by themselves had noeffect on HDL function when given together acted synergistically. WhenD-4F and a statin were given together to apo E null mice, theirpro-inflammatory HDL at 50 μg/ml of HDL-cholesterol became as effectiveas normal human HDL at 350 μg/ml of HDL-cholesterol in preventing theinflammatory response induced by the action of HPODE oxidizing PAPC incocultures of human artery wall cells.

Example 3 Use of Orally Administered Peptides to Ameliorate Osteoporosis

D-4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂, SEQ ID NO: 5) at 1mg/ml was or was not added to the drinking water of apoE null mice (8mice per group). After 6 weeks mice were euthanized and the left femurfrom each mouse removed and analyzed by quantitative CT scanning todetermine BMD. Scans were performed at 4 longitudinal axis positions(slices) for each femur, with 1 being most distal and 4 most proximal.Values of BMD are expressed as mean±SEM.

It was found that adding D-4F to the drinking water of apoE null micefor 6 weeks dramatically increased trabecular bone mineral density(Table 3).

TABLE 3 Effect of D-4F on bone mineral density (BMD) in mice. TrabecularBone Mineral Density (mg/cm³) Slice Water D-4F Fold Increase 1 3.1 ± 318 ± 4 5.8 2 1.9 ± 2 12 ± 6 6.3 3 1.7 ± 1 11 ± 6 6.5 4 7.3 ± 4 16 ± 62.2

It is interesting that bisphosphonates are particularly active ontrabecular bone (Bohic et al. (2000) Bone, 26: 341-348; Ramamurthy etal. (2001) Curr Med Chem., 8: 295-303; Rohanizadeh et al. (2000) CalcifTissue Int., 67: 330-336; Rodan (1997) Bone 20:1-4). Since it wasdetermined that D-4F did not alter the lipid profile in these mice, itis likely that the beneficial effects of the peptide are due tointerference with the inhibitory actions of oxidized lipids on bone. Ourdata cannot differentiate between an action on osteoblasts orosteoclasts but these preliminary data strongly suggest that D-4F may bean excellent agent to inhibit/prevent/treat osteoporosis.

Example 4 “L” Form Peptides are Effective

While the peptides of this invention, when synthesized from D-aminoacids, were more effective when given orally than peptides comprisingall L-amino acids (“L-form peptides”), the L-form peptides were alsoeffective. This is amply illustrated in the examples provided in thepriority documents of the present application (e.g. U.S. Ser. Nos.09/645,454, 09/896,841, 10/187,215, and 10/273,386) all of which areincorporated herein by reference. For example in Example 1 on pages 46to 57 of U.S. Ser. No. 10/187,215 (the '215 application), thespecification of which is published in PCT/US01/26497 (WO 02/15923),evidence was presented that the peptide known as 5F synthesized fromL-amino acids (Sequence ID No 6 in Table 1 page 27) when given byinjection was cleared from the circulation of the mouse with a T_(1/2)of 6.22 hours (see Table 3 on page 51), was not toxic (see page 52 andTable 4 on page 52 of the '215 application), was not antigenic (see topof page 53), and dramatically improved the ability of the mouse HDL toinhibit the oxidation of LDL and prevent LDL-induced monocytechemotactic activity in human artery wall cell cocultures (see page 54and FIG. 7). Additionally as shown at the bottom of page 54 and in FIG.8 of the '215 application, the injection of the 5F peptide dramaticallyreduced atherosclerotic lesions in the mice.

In Example 3 of the '215 application at the bottom of page 73 and inFIG. 18 we demonstrated that incubation of 4F, 5F and 6F, allsynthesized from L-amino acids dramatically reduced LDL-induced monocytechemotactic activity in a human artery wall coculture.

In Example 5 of the '215 application, as indicated at the bottom of page78 and in FIG. 22A, the peptide, 4F, when given by mouth to mice wasmore degraded when it was synthesized from L-amino acids than was thecase when the peptide was synthesized from D-amino acids. However asshown in FIG. 22A some intact peptide was found in the circulation afteroral administration of L-4F. Furthermore, as shown in FIG. 22B the mouseHDL was improved after oral administration of L-4F in terms of itsability to inhibit monocyte chemotactic activity when human artery wallcells were exposed to human LDL, although the improvement in HDL'santi-inflammatory properties were not as dramatic as when the peptidewas synthesized from all D-amino acids.

In summary, the examples detailed in the priority documents of thisapplication and incorporated herein by reference demonstrate thatadministration of the peptides of this invention were effective whethersynthesized from L or D-amino acids, although the D-form peptides weremore effective when given orally.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of mitigating one or more symptoms of scleroderma in amammal, said method comprising administering to said mammal an effectiveamount of a peptide or a concatamer of a peptide that: ranges in lengthup to about 30 amino acids; comprises the amino acid sequenceD-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ ID NO: 5); and bears at leastone protecting group.
 2. The method of claim 1, wherein said peptide isin a pharmaceutically acceptable excipient.
 3. The method of claim 1,wherein said peptide is in a pharmaceutically acceptable excipientsuitable for oral administration.
 4. The method of claim 1, wherein saidpeptide is administered as a unit dosage formulation.
 5. The method ofclaim 1, wherein said administering comprises administering said peptideby a route selected from the group consisting of oral administration,nasal administration, rectal administration, intraperitoneal injection,intravascular injection, subcutaneous injection, transcutaneousadministration, and intramuscular injection.
 6. The method of claim 1,wherein said mammal is a mammal diagnosed as having one or more symptomsof scleroderma.
 7. The method of claim 1, wherein said mammal is amammal diagnosed as at risk for stroke or atherosclerosis.
 8. The methodof claim 1, wherein said mammal is a human.
 9. The method of claim 1,wherein said mammal is non-human mammal.
 10. The method of claim 1,wherein said peptide is s formulated as a dry powder.
 11. The method ofclaim 1, wherein said peptide is formulated in a sterile excipient. 12.The method of claim 1, wherein said peptide consists of all “D” aminoacids.
 13. The method of claim 1, wherein said peptide consists of all“L” amino acids.
 14. The method according to any of claims 12, and 13,wherein the amino acid sequence of said peptide consists of the sequenceD-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F (SEQ ID NO: 5).
 15. The method ofclaim 14, wherein said peptide comprises a first protecting groupcoupled to the amino terminus and/or a second protecting group coupledto the carboxyl terminus.
 16. The method of claim 15, wherein said firstprotecting group and/or said second protecting group, when present, areindependently selected from the group consisting of acetyl (Ac), amide,a 3 to 20 carbon alkyl group, Fmoc, t-butoxycarbonyl (Tboc),9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-fluorenecarboxylicgroup, 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl(Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt),4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl(Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl(MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl(2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl(tBu), and trifluoroacetyl (TFA).
 17. The method of claim 14, whereinsaid peptide comprises a first protecting group coupled to the aminoterminus and said amino terminal protecting group is a protecting groupselected from the group consisting of a benzoyl, an acetyl, a propionyl,a carbobenzoxy, a propyl, a butyl, a pentyl, a hexyl, an N-methylanthranilyl, and a 3 to 20 carbon alkyl.
 18. The method of claim 14,wherein said peptide comprises a protecting group coupled to thecarboxyl terminus and said carboxyl terminal protecting group is anamide.
 19. The method of claim 18, wherein said peptide has the formulaAc-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F—NH₂ (SEQ ID NO: 5).
 20. Themethod of claim 19, wherein said peptide is formulated as a unit dosageformulation.